Interleukin-2 variants and their use

Abstract

The present invention relates to novel interleukin-2 (IL-2) muteins. The present invention further provides fusion proteins, dimeric molecules, and immunoconjugates containing the IL-2 muteins, as well as nucleic acids encoding them, vectors and host cells containing the nucleic acids, and pharmaceutical compositions and therapeutic uses comprising them. The present invention also provides methods for preparing the IL-2 muteins, fusion proteins, dimeric molecules, and immunoconjugates.

Description

[Technical Field]

[0001] This invention relates to a novel interleukin-2 (IL-2) mutant protein and its use. Specifically, this invention relates to an IL-2 mutant protein having improved properties compared to wild-type IL-2, such as improved IL-2 receptor binding properties and improved drug potential. This invention further provides a fusion protein, dimer, immune complex containing the IL-2 mutant protein, and nucleic acids encoding the IL-2 mutant protein, dimer, immune complex, vectors containing the nucleic acids, and host cells. This invention further provides methods for preparing the IL-2 mutant protein, fusion protein, dimer, and immune complex, pharmaceutical compositions containing the same, and therapeutic uses. [Background technology]

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), primarily affects activated T cells, especially CD4 cells. + IL-2 is a pluripotent cytokine 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 the removal of the 20 amino acids at the N-terminus, it produces mature secreted IL-2. Sequences of IL-2 from other species have also been disclosed; see NCBI Ref Seq No. NP032392 (mouse), NP446288 (rat), or NP517425 (chimpanzee).

[0003] Interleukin-2 possesses four antiparallel amphiphilic 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 acts via 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). While IL-2Rβ and IL-2Rγ are crucial for IL-2 signaling, IL-2Rα (CD25) is not essential for signaling but can provide high-affinity binding of IL-2 to its receptor (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimer receptor formed by combining IL-2Rα, β, and γ (IL-2αβγ) is a high-affinity IL-2 receptor (KD of approximately 10 pM), the dimer receptor consisting of β and γ (IL-2βγ) is an intermediate-affinity receptor (KD of approximately 1 nM), and the IL-2 receptor formed by only the α subunit is a low-affinity receptor.

[0004] Immune cells express dimeric or trimer IL-2 receptors. Dimeric receptors express cytotoxic CD8 + It is expressed in T cells and natural killer (NK) cells, and the trimer receptor is mainly expressed on activated lymphocytes and CD4 + CD25 + FoxP3 + It is expressed in inhibitory regulatory T cells (Tregs) (Byman, O. and Sprent, J. Nat. Rev. Immunol. 12, 180-190 (2012)). Quiescing effector T cells and NK cells do not have 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 vivo, so IL-2 usually preferentially stimulates the proliferation of Treg cells.

[0005] IL-2 mediates multiple effects in the immune response through binding to IL-2 receptors on different cells. On the one hand, IL-2 has an immune system stimulating effect, and can stimulate the proliferation and differentiation of T cells and natural killer (NK) cells. Therefore, IL-2 is approved for use as an immunotherapeutic agent in the treatment of cancer and chronic viral infections. On the other hand, IL-2 is also involved in immunosuppressive CD4 + CD25 + It can also 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)), and induce immunosuppression by activated Treg cells in patients.

[0006] Furthermore, years of clinical experience have shown that while high doses of IL-2 can produce significant clinical effects in the treatment of cancers such as melanoma and kidney cancer, they can also cause severe drug-related toxicity and side effects, including cardiovascular toxicity such as vascular leak syndrome and hypotension. Studies have indicated that these toxicities are likely caused by excessive activation of lymphocytes (particularly T cells and NK cells) by IL-2, which stimulates the release of inflammatory factors. For example, this can constrict vascular endothelial cells, increase the gaps between cells, cause leakage of tissue fluid, and thereby lead to the side effect of vascular leak syndrome.

[0007] Another limiting issue regarding the clinical use of IL-2 is the difficulty of administration due to its very short half-life. Because IL-2 has a molecular weight of only 15 kDa, it is primarily removed by glomerular filtration, resulting in a human half-life of only about one hour. Clinically, high doses of IL-2 need to be infused every eight hours to achieve a sufficiently high human exposure. However, frequent administration not only places a significant burden on patients, but more importantly, high-dose infusions of IL-2 cause very high peak blood concentrations (Cmax), which is likely another factor contributing to drug toxicity. Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8:15373, DOI:10.1038 / ncomms15373) proposed improving the pharmacodynamic properties of interleukins by preparing interleukin-2-Fc fusions, but these fusion proteins are expressed at low levels and tend to form aggregates.

[0008] In the production of IL-2, the natural IL-2 molecule is extremely difficult to express in mammalian cells (CHO or HEK293) due to the characteristics of its amino acid sequence, and its molecular stability is also relatively poor. Therefore, the currently commercially approved IL-2 molecule, Proleukin, is produced in a prokaryotic bacterial expression system. However, IL-2-Fc fusion proteins cannot be expressed in bacterial systems. Therefore, in this field, there is a need to improve the expression of IL-2 and IL-2-Fc molecules in mammalian cells.

[0009] In this field, methods for manipulating the IL-2 molecule have been proposed. For example, Helen R. Mott et al. have disclosed the mutant human IL-2 protein F42A, which has excluded IL-2Rα binding ability. Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8:15373, DOI:10.1038 / ncomms15373) have disclosed the triple mutant human IL-2 mutant protein IL-2, which has excluded IL-2Rα binding ability. 3XIt has also been proposed that the protein has residue mutations R38D+K43E+E61R at amino acid residue positions 38, 43, and 61, respectively. CN1309705A discloses mutations at positions D20, N88, and Q126, which result in reduced binding between IL-2 and IL-2Rβγ. These mutant proteins still have defects in pharmacokinetic and / or pharmacodynamic properties, and when expressed in mammalian cells, they also have problems with low expression levels and / or relatively poor molecular stability.

[0010] Given the aforementioned issues related to IL-2 immunotherapy and production, the field still needs to develop novel IL-2 molecules with improved properties, particularly those useful in production and purification, and those with improved pharmacokinetic and pharmacodynamic characteristics. [Overview of the project]

[0011] The present invention satisfies the above requirements by providing a long-acting IL-2 mutant protein molecule having improved drug potential and / or improved IL-2 receptor binding properties.

[0012] Accordingly, in one embodiment, the present invention provides a novel IL-2 mutant protein. In some embodiments, the IL-2 mutant protein of the present invention has one or more of the following properties, preferably at least properties (i) and (ii): (i) Improved drug potential, particularly improved expression levels and / or purification performance when expressed in mammalian cells, (ii) Weakening of binding with IL-2Rβγ, (iii) Reduction or elimination of binding to IL-2Rα.

[0013] In some embodiments, the IL-2 mutant protein of the present invention has characteristics (i) and (ii) and maintains binding to IL-2Rα compared to the wild-type IL-2 protein. In some other embodiments, the IL-2 mutant protein of the present invention has characteristics (i) to (iii).

[0014] In some embodiments, the present invention provides an IL-2 mutant protein comprising a mutation at the IL-2Rβγ binding interface and further comprising a mutation at the IL-2Rα binding interface and / or a shortened B'C' loop region. In some preferred embodiments, the present invention provides an IL-2 mutant protein comprising a mutation at the IL-2Rβγ binding interface and a shortened B'C' loop region, but not comprising a mutation at the IL-2Rα binding interface.

[0015] Furthermore, the present invention provides a fusion protein, dimer protein, immune complex, pharmaceutical composition and combination product containing an IL-2 mutant protein, an encoding nucleic acid, a vector and host cell containing the nucleic acid, and a method for producing the IL-2 mutant protein, fusion protein, dimer protein and immune complex of the present invention.

[0016] Furthermore, the present invention also provides methods for treating diseases using the IL-2 mutant protein, fusion protein, dimeric protein, and immune complex of the present invention, as well as methods and uses for stimulating the immune system of a subject.

[0017] The present invention will be further described below with reference to the drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the invention, and modifications that are readily conceivable to those skilled in the art are included in the spirit of the invention and the appended claims. [Brief explanation of the drawing]

[0018] [Figure 1] The crystal structure (PDB:1Z92) of the IL-2 and IL-2Rα composite is shown. [Figure 2A] The crystal structure of IL-2 (PBD:2ERJ) (A) and the B'C' loop structures of human IL-2 and human IL-15 (B) are shown. [Figure 2B]The crystal structure of IL-2 (PBD:2ERJ) (A) and the B'C' loop structures of human IL-2 and human IL-15 (B) are shown. [Figure 3] This shows IL-2 mutant proteins and their sequences screened from the mutant library IBYDL029. [Figure 4] The crystal structures (PBD:2ERJ) of IL-2 and IL-2Rβγ and their contact interface are shown. [Figure 5] A schematic molecular diagram of the IL-2-Fc dimeric protein is shown. [Figure 6] The structures of some exemplary IL-2 weakening molecules of the present invention are shown. [Figure 7A] The selected and constructed IL-2mutant-Fc dimer protein shows signal curves activating p-STAT5 in normal, unactivated T lymphocytes (CD4+ T cells and CD8+ T cells (AE)) and activated T lymphocytes (CD4+CD25+ T cells (F)). [Figure 7B] The selected and constructed IL-2mutant-Fc dimer protein shows signal curves activating p-STAT5 in normal, unactivated T lymphocytes (CD4+ T cells and CD8+ T cells (AE)) and activated T lymphocytes (CD4+CD25+ T cells (F)). [Figure 7C] The selected and constructed IL-2mutant-Fc dimer protein shows signal curves activating p-STAT5 in normal, unactivated T lymphocytes (CD4+ T cells and CD8+ T cells (AE)) and activated T lymphocytes (CD4+CD25+ T cells (F)). [Figure 7D] The selected and constructed IL-2mutant-Fc dimer protein shows signal curves activating p-STAT5 in normal, unactivated T lymphocytes (CD4+ T cells and CD8+ T cells (AE)) and activated T lymphocytes (CD4+CD25+ T cells (F)). [Figure 7E]The selected and constructed IL-2mutant-Fc dimer protein shows signal curves activating p-STAT5 in normal, unactivated T lymphocytes (CD4+ T cells and CD8+ T cells (AE)) and activated T lymphocytes (CD4+CD25+ T cells (F)). [Figure 7F] The selected and constructed IL-2mutant-Fc dimer protein shows signal curves activating p-STAT5 in normal, unactivated T lymphocytes (CD4+ T cells and CD8+ T cells (AE)) and activated T lymphocytes (CD4+CD25+ T cells (F)). [Figure 8A] Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 8B] Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 8C] Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 8D] Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 8E] Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 8F] Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 8G]Compared to the control rhIL-2 protein, the weakened IL-2 mutant-Fc dimer protein shows signaling curves that activate p-STAT5 in different lymphocyte subsets. [Figure 9A] This shows the effects of IL-2-Fc dimer protein, constructed from a weakened IL-2 mutant molecule with reduced CD25 binding affinity, on tumor volume (A) and on animal body weight and body weight change (B and C) when administered to tumor-bearing C57 mice. [Figure 9B] This shows the effects of IL-2-Fc dimer protein, constructed from a weakened IL-2 mutant molecule with reduced CD25 binding affinity, on tumor volume (A) and on animal body weight and body weight change (B and C) when administered to tumor-bearing C57 mice. [Figure 9C] This shows the effects of IL-2-Fc dimer protein, constructed from a weakened IL-2 mutant molecule with reduced CD25 binding affinity, on tumor volume (A) and on animal body weight and body weight change (B and C) when administered to tumor-bearing C57 mice. [Figure 10A] This shows the effects of IL-2-Fc dimer protein, constructed from a weakened IL-2 mutant molecule with maintained CD25 binding affinity, on tumor volume (A) and on animal body weight and body weight change (B and C) when administered to tumor-bearing C57 mice. [Figure 10B] This shows the effects of IL-2-Fc dimer protein, constructed from a weakened IL-2 mutant molecule with maintained CD25 binding affinity, on tumor volume (A) and on animal body weight and body weight change (B and C) when administered to tumor-bearing C57 mice. [Figure 10C] This shows the effects of IL-2-Fc dimer protein, constructed from a weakened IL-2 mutant molecule with maintained CD25 binding affinity, on tumor volume (A) and on animal body weight and body weight change (B and C) when administered to tumor-bearing C57 mice. [Figure 11A]The results of detecting body weight and body weight change in mice before administration of IL-2mutantFc dimer protein and on days 3 and 7 after administration (A and B), as well as the results of detecting Treg, NK, CD4+, and CD8+ T cells in the blood (CF), are shown. [Figure 11B] The results of detecting body weight and body weight change in mice before administration of IL-2mutantFc dimer protein and on days 3 and 7 after administration (A and B), as well as the results of detecting Treg, NK, CD4+, and CD8+ T cells in the blood (CF), are shown. [Figure 11C] The results of detecting body weight and body weight change in mice before administration of IL-2mutantFc dimer protein and on days 3 and 7 after administration (A and B), as well as the results of detecting Treg, NK, CD4+, and CD8+ T cells in the blood (CF), are shown. [Figure 11D] The results of detecting body weight and body weight change in mice before administration of IL-2mutantFc dimer protein and on days 3 and 7 after administration (A and B), as well as the results of detecting Treg, NK, CD4+, and CD8+ T cells in the blood (CF), are shown. [Figure 11E] The results of detecting body weight and body weight change in mice before administration of IL-2mutantFc dimer protein and on days 3 and 7 after administration (A and B), as well as the results of detecting Treg, NK, CD4+, and CD8+ T cells in the blood (CF), are shown. [Figure 11F] The results of detecting body weight and body weight change in mice before administration of IL-2mutantFc dimer protein and on days 3 and 7 after administration (A and B), as well as the results of detecting Treg, NK, CD4+, and CD8+ T cells in the blood (CF), are shown. [Figure 12] This shows the amino acid sequence of the wild-type IL-2 protein IL-2WT (SEQ ID NO: 1), its amino acid residue numbering, and its sequence alignment with the mutant protein IL-23X. [Modes for carrying out the invention]

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. For the purposes of this invention, the following terms are defined below.

[0020] When used with a number or figure, the term "approximately" means covering a range of numbers or figures that is 5% smaller than the lower limit and 5% larger than the upper limit.

[0021] The term "and / or" should be understood to refer to any one of the selectable options, or any combination of any two or more selectable options.

[0022] As used herein, the terms “inclusive” or “contains” mean including the elements, integers, or steps described, but not excluding any other elements, integers, or steps. Wherever the terms “inclusive” or “contains” are used herein, unless otherwise specified, this also includes the elements, integers, or steps mentioned. For example, when referring to an IL-2 mutant protein that “inclusive” or “contains” a certain mutation or combination of mutations, it is also intended to include IL-2 mutant proteins having only that mutation or combination of mutations.

[0023] In this specification, wild-type “interleukin-2” or “IL-2” refers to the parental IL-2 protein, preferably a naturally occurring IL-2 protein, as a template for introducing the mutation or combination of mutations of the present invention, including, for example, the untreated (e.g., signal peptide not removed) and treated (e.g., signal peptide removed) form, naturally occurring IL-2 proteins from humans, mice, rats, and non-human primates. The full-length sequence of naturally occurring human IL-2 including the signal peptide is shown in SEQ ID NO: 2, and the sequence of its mature protein is shown in SEQ ID NO: 3. This expression also includes naturally occurring IL-2 allele variants and splice variants, isotypes, homologs, and species homologs. This expression also includes variants of natural IL-2, for example, which may have at least 95% to 99% or more identity with natural IL-2, or may have 1 to 10 or 1 to 5 amino acid mutations (e.g., conservative substitutions), and preferably have essentially the same IL-2Rα binding affinity and / or IL2Rβγ binding affinity as the natural IL-2 protein. Therefore, in some embodiments, wild-type IL-2 may contain amino acid mutations that do not affect its binding to the IL-2 receptor compared to the natural IL-2 protein, for example, the natural human IL-2 protein with the mutation C125S introduced at position 125 (uniprot:P60568) belongs to the wild-type IL-2 of the present invention. One example of a wild-type human IL-2 protein containing the C125S mutation is shown in SEQ ID NO: 1. In some embodiments, the wild-type IL-2 sequence has at least 85%, 95%, and even more than 96%, 97%, 98%, or 99% or higher amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0024] In the present specification, the amino acid mutations may be amino acid substitutions, deletions, insertions, and additions. By performing any combination of substitutions, deletions, insertions, and additions, a final mutant protein construct having desired properties (e.g., reduced IL-2Rα binding affinity and / or improved drug discovery potential and / or attenuated IL-2Rβγ) can be obtained. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy termini of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. For example, the length of the loop region can be shortened by deleting an alanine residue at position 1 of full-length human IL-2 or deleting one or more amino acids in the B’C’ loop region. In some embodiments, preferred amino acid mutations are amino acid substitutions, such as combinations of single amino acid substitutions or substitutions of amino acid sequence segments. For example, the whole or part of the B’C’ loop region sequence of wild-type IL-2 can be replaced with a different sequence, preferably resulting in a B’C’ loop region sequence with a shortened length.

[0025] In the present invention, when referring to an amino acid position in an IL-2 protein or an IL-2 sequence segment, the wild-type human IL-2 protein (IL-2 WTThe amino acid position is determined by referring to the amino acid sequence SEQ ID NO: 1 of SEQ ID NO: (also known as IL-2) (shown in Figure 8). By aligning the amino acid sequence with SEQ ID NO: 1, the corresponding amino acid position on other IL-2 proteins or polypeptides (including full-length sequences or cleavage fragments) can be identified. Therefore, in this invention, unless otherwise specified, the amino acid position of an IL-2 protein or polypeptide is an amino acid position numbered according to SEQ ID NO: 1. For example, when referring to "F42", it refers to the phenylalanine residue F at position 42 of SEQ ID NO: 1, or the amino acid residue at the corresponding position aligned on other IL-2 polypeptide sequences. Sequence alignment performed for amino acid position determination can be done using the Basic Local Alignment Search Tool, which can be obtained from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0026] In this specification, when referring to IL-2 mutant proteins, single amino acid substitutions are described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, a substitution of lysine at position 35 by glutamate can be represented as K35E. If there are multiple selectable amino acid substitution schemes (e.g., D, E) at a given position (e.g., K35), the amino acid substitution can be represented as K35D / E. Correspondingly, individual single amino acid substitutions can be linked by a plus sign "+" or "-" to represent combination mutations at multiple given positions. For example, a combination mutation of positions K35E, T37E, R38E, and F42A can be represented as K35E+T37E+R38E+F42A, or K35E-T37E-R38E-F42A.

[0027] In this specification, the “sequence identity percentage” 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., Sequence ID 1). Sequence alignment methods for comparison are well known in the art. Algorithms applied to determine the sequence identity percentage include, for example, the BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25:3389-402, 1977 and Altschul et al. J. Mol. Biol. 215:403-10, 1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. For the purposes of this application, the identity percentage can be determined using default parameters with the Basic Local Alignment Search Tool, which can be obtained from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0028] As used herein, the term “conservative substitution” means an amino acid substitution that does not adversely affect or alter the biological function of a protein / polypeptide, including its 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 substitution of one amino acid with another amino acid having similar chemical properties (e.g., charge or hydrophobicity). A table of conservative substitutions of functionally similar amino acids is well known in the art. In the present invention, the conservative substitution residues are derived from the following Conservative Substitution Table X, in particular from preferred conservative amino acid substitution residues in Table X.

[0029] [Table 1]

[0030] For example, the wild-type IL-2 protein may have a conserved amino acid substitution for one of SEQ ID NOs: 1-3, or may have only a conserved amino acid substitution, and in one preferred embodiment, the conserved substitution has 10 or fewer amino acid residues, such as residues 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, for example, the mutant IL-2 protein of the present invention may have a conserved amino acid substitution for any of the IL-2 mutant protein sequences specifically shown herein (e.g., any one of SEQ ID NOs: 37-638), or may have only a conserved amino acid substitution, and in one preferred embodiment, the conserved substitution has 10 or fewer amino acid residues, such as residues 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0031] "Affinity" or "binding affinity" can be used to reflect the internal bonding ability of the interaction between members of a binding pair. The affinity of molecule X for its binding partner Y is expressed by the equilibrium dissociation constant (K D The equilibrium dissociation constant may also be expressed by the dissociation rate constant and the coupling rate constant (k dis and k on It is the ratio of ( ). Binding affinity may be measured by commonly used methods known in the art. One specific method for measuring affinity is the ForteBio affinity measurement technique described herein.

[0032] In this specification, the antigen-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 a Fab fragment and an scFv fragment.

[0033] In this specification, an antibody Fc fragment is the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region, and may include native sequence Fc fragments and mutant Fc fragments. Native sequence Fc fragments include various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al, IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 or Pro230 of the heavy chain to the carboxyl group terminus. In other embodiments, the C-terminal lysine (Lys447) of the Fc fragment may or may not be present. In some other embodiments, the Fc fragment is a mutant Fc fragment containing a mutation, such as the L234A-L235A mutation. Unless otherwise specified herein, the numbering of amino acid residues in Fc fragments is according to the EU numbering system, also known as the EU index. For example, see Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. In some embodiments, the antibody Fc fragment may have an IgG1 hinge sequence or a portion of an IgG1 hinge sequence at its N-terminus, for example, sequences E216 to T225 or D221 to T225 according to the EU numbering system. Mutations may be present in the hinge sequence.

[0034] The IL-2 protein belongs to the family of short-chain type I cytokines, which have a four-α-helix bundle structure (A, B, C, D). In this specification, the terms “B'C'Loop,” “B'C'Loop Region,” or “B'C'Loop Sequence” may be used interchangeably and refer to the linking sequence between the B helix and C helix of the IL-2 protein. The B'C' loop sequence of a single IL-2 protein can be determined by analysis of the crystal structure of IL-2 (e.g., PDB:2ERJ). For the purposes of this invention, according to the numbering in SEQ ID NO: 1, the B'C' loop sequence refers to a sequence linking the residues at position 72 and position 84 in the IL-2 polypeptide. In the wild-type IL-2 proteins of SEQ ID NOs: 1, 2, and 3, the linking sequence contains a total of 11 amino acids, A73-R83. Correspondingly, in this specification, the terms “shortened loop region” or “shortened B'C' loop region” refer to a mutant protein having a shortened B'C' loop sequence compared to the wild-type IL-2 protein, i.e., according to the numbering in Sequence ID No. 1, the length of the linkage sequence between amino acid residues aa72 and aa84 is shortened. The “shortened loop region” can be achieved by substitution or cleavage of the loop sequence. The substitution or cleavage can occur in any region or portion of the B'C' loop sequence. For example, the substitution or cleavage may be a substitution of the loop region A73-R83 sequence or a cleavage from one or more amino acid residues at the C-terminus of said sequence. Furthermore, for example, the substitution or cleavage may be a substitution of the loop region Q74-R83 sequence or a cleavage from one or more amino acid residues at the C-terminus of said sequence. After the substitution or cleavage, if necessary, single amino acid substitutions, such as amino acid substitutions to eliminate glycosylation, and / or reverse mutations can be further introduced into the loop region sequence to further improve the properties of the mutant protein, such as drug potential. Therefore, in this specification, the shortened B'C' loop region after mutation can be described by concatenating the sequence between the residue at position 72 and the residue at position 84 after the introduction of the mutation.

[0035] In this specification, “IL-2Rα binding interface” mutations refer to mutations occurring at amino acid sites where IL-2 and IL-2Rα (i.e., CD25) interact. These interaction sites can be determined by analyzing the crystal structure of IL-2 and its receptor complex (e.g., PDB:1Z92). In some embodiments, the mutations refer specifically to mutations in the amino acid residue 35-72 region of IL-2, and more specifically to mutations at amino acid sites 35, 37, 38, 41, 42, 43, 45, 61, 62, 68, and 72. Preferably, the IL-2 protein containing the mutation has reduced or eliminated IL-2Rα binding compared to the corresponding protein before the introduction of the mutation.

[0036] In this specification, “IL-2βγ binding interface” mutations refer to mutations occurring at amino acid sites where IL-2 and IL-2Rβγ (i.e., CD122 and CD132) interact. These interaction sites can be determined by analyzing the crystal structure of IL-2 and its receptor complex (e.g., PDB:2ERJ). In some embodiments, the mutations refer particularly to mutations in the amino acid residues 12-20, 84-95, and generally the 126 region of IL-2, and more specifically to mutations at amino acid sites 12, 15, 16, 19, 20, 84, 87, 88, 91, 92, 95, and 126. Preferably, the IL-2 protein containing the mutation has a weakened IL-2Rβγ binding compared to the corresponding protein before the introduction of the mutation.

[0037] In this specification, with respect to IL-2Rβγ receptor binding, "weakening" of the IL-2 protein molecule refers to introducing a mutation at the IL-2Rβγ binding interface, where the mutation results in a reduction in binding affinity to the IL-2Rβγ receptor compared to the corresponding IL-2 protein before the introduction of the mutation. More preferably, compared to the corresponding protein, the weakened molecule has reduced activity to activate T cells (e.g., CD25-T cells or CD25+ T cells) and / or NK cells. For example, by detecting the ratio of the EC50 values ​​that activate the T cell pSTAT5 signal of the weakened molecule and the corresponding protein, the reduction can reach, for example, 5 times or more, for example, 10 times or more, or 50 times or more, or 100 times or more, or even 1000 times or more. For example, compared to the corresponding protein, the T cell activating activity of the weakened molecule can be reduced by a factor of 10 to 50 times, or 50 to 100 times, or 100 to 1000 times, or even more. Therefore, in some embodiments of the present invention, the weakening molecule of the present invention has a "weakened" binding affinity to the IL-2Rβγ receptor and "weakened" T cell activation activity.

[0038] Various aspects of the present invention will be described in further detail in the following sections.

[0039] 1. The IL-2 mutant protein of the present invention

[0040] Advantageous biological properties of the IL-2 mutant protein of the present invention

[0041] Through long-term research, the inventors have discovered that by combining the following molecular mutations and modifications, it is possible to simultaneously improve the efficacy of IL-2, reduce its toxicity and side effects, and achieve good production performance.

[0042] (i) By introducing specific residue mutations at the binding interface of IL-2 to the IL-2Rβγ receptor, binding to the IL-2Rβγ receptor is weakened, and the activity of IL-2 is downregulated to some extent. By including such mutations that weaken IL-2Rβγ receptor binding, the IL-2 mutant protein of the present invention can activate lymphocytes to kill tumors while simultaneously avoiding the release of large amounts of inflammatory factors caused by excessive lymphocyte activation and the resulting drug-related toxicity. Furthermore, these weakening mutations reduce the binding affinity of the IL-2 mutant protein of the present invention to the IL-2 receptor, which is widely present on lymphocytes, thereby reducing the clearance of the IL-2 mutant protein mediated by the IL-2 receptor and delaying the duration of action of the IL-2 mutant protein.

[0043] (ii) Construct the mutant IL-2 protein of the present invention into an IL-2-Fc dimer. The formation of this dimer increases the molecular weight of the mutant IL-2 protein of the present invention, significantly reduces renal clearance, and further extends the half-life of the IL2-Fc fusion protein through in vivo FcRn-mediated recycling. This overcomes the problems of the short half-life of IL-2 and the high peak blood concentration caused by high-frequency and high-dose administration.

[0044] (iii) Modify the B'C' loop structure of IL-2, for example by substituting it with the loop of an IL-15 molecule or by cleaving the B'C' loop of the IL-2 molecule. Such B'C' loop mutations significantly enhance the stability of the B'C' loop structure in the IL-2 mutant protein of the present invention and can significantly improve the production performance of the IL-2 mutant protein and the IL-2-Fc dimer molecule constructed therefrom, such as significantly improved expression levels and purity.

[0045] Furthermore, the inventors have found that, in the mutant protein of the present invention, while maintaining the above-mentioned excellent properties, the binding activity of the IL-2 mutant protein to IL-2Rα can be modified as needed to meet the different drug formation requirements of IL-2 in many aspects, such as antitumor or autoimmune disease treatment, thereby further conferring excellent pharmacodynamic properties to the mutant protein of the present invention.

[0046] For example, the mutant protein of the present invention may retain substantially equivalent IL-2Rα binding activity to wild-type IL-2, or (iv) one or more specific mutations at the binding interface of IL-2 to the IL-2Rα receptor may be combined to alter the binding performance of the IL-2 mutant protein to IL-2Rα.

[0047] As a result, by modifying the sequence, the IL2-Fc series molecules of the present invention have their binding affinity to the receptor IL2Rβ / γ weakened, achieving superior pharmacokinetic experimental results and efficacy results, while significantly improving drug discovery potential in terms of protein expression level and purity.

[0048] Therefore, the present invention provides an IL-2 mutant protein having improved drug potential and improved IL-2 receptor binding properties. The IL-2-Fc molecule containing the IL-2 mutant protein of the present invention can effectively avoid the excessive release of inflammatory factors caused by strong stimulation of lymphocytes and has more stable and longer-acting pharmacokinetic properties. Therefore, a sufficiently high human drug exposure can be achieved with a relatively low single dose to avoid drug-related toxicity due to excessively high Cmax. More significantly, although the long-acting IL-2-Fc molecule of the present invention has weakened lymphocyte immunostimulatory activity compared to natural IL-2, its pharmacokinetic properties are significantly improved. As a result, the duration of the in vivo effective drug concentration of the molecule of the present invention is longer, exerting relatively long-lasting continuous stimulation of lymphocytes, achieving pharmacodynamic effects equivalent to or better than those of natural IL-2 molecules, and achieving superior antitumor effects and resistance in animals.

[0049] Improved drug discovery potential

[0050] In some embodiments, the IL-2 mutant proteins of the present invention have improved drug potential. For example, when expressed in mammalian cells such as HEK293 or CHO cells, and particularly when expressed in Fc fusion proteins, they have one or more of the following characteristics: (i) higher expression levels than wild-type IL-2 protein, and (ii) easier purification to higher protein purity.

[0051] In some embodiments of the present invention, the IL-2 mutant protein of the present invention exhibits increased expression levels compared to wild-type IL-2. In some embodiments of the present invention, the increase in expression occurs in mammalian cell expression systems. The expression level can be measured by any suitable method that allows for quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in cell culture supernatant (preferably supernatant after one-step affinity chromatography purification). For example, the amount of recombinant IL-2 protein in a sample can be assessed by Western blotting or ELISA. In some embodiments, the IL-2 mutant protein of the present invention exhibits increased expression levels in mammalian cells by at least 1.1 times, or at least 1.5 times, or at least 2 times, 3 times or 4 times or more, or at least 5, 6, 7, 8 or 9 times, or even more than 10 times, 15, 20, 25, 30 and 35 times, etc., compared to wild-type IL-2.

[0052] As demonstrated by measuring the purity of the purified protein after protein A affinity chromatography in some embodiments, the IL-2 mutant protein-Fc fusion of the present invention exhibits higher purity compared to the wild-type IL-2 protein fusion. In some embodiments, protein purity is detected by SEC-HPLC technique. In some preferred embodiments, after one-step protein A affinity chromatography purification, the purity of the IL-2 mutant protein-Fc fusion of the present invention can reach 70%, 80%, or 90% or higher, preferably 92%, 93%, 94%, 95%, 98%, or 99% or higher.

[0053] In some embodiments, as demonstrated by measuring the purity of the purified protein after protein A affinity chromatography, the IL-2-Fc dimer protein of the present invention exhibits higher purity compared to the corresponding IL-2-Fc dimer protein formed by wild-type IL-2 protein. In some embodiments, protein purity is detected by SEC-HPLC technique. In some preferred embodiments, after one-step protein A affinity chromatography purification, the purity of the IL-2-Fc dimer protein of the present invention can reach 70%, 80%, or 90% or higher, preferably 92%, 93%, 94%, 95%, 98%, or 99% or higher.

[0054] Weakened IL-2βγ receptor binding

[0055] In some embodiments, by introducing a mutation at the IL-2Rβγ binding interface, the IL-2 mutant protein of the present invention has a weakened IL-2βγ binding affinity compared to the corresponding protein before the introduction of the mutation.

[0056] In some embodiments, the IL-2 mutant protein of the present invention exhibits reduced binding affinity to IL-2Rβ and / or IL-2Rβγ receptors compared to the protein before attenuation by introducing mutations at the IL-2Rβγ binding interface. In some embodiments, the IL-2 mutant protein of the present invention exhibits reduced binding affinity to the IL-2Rβ receptor compared to the protein before attenuation, reduced by 1 to 20 times or more, including, for example, the removal of binding to the IL-2Rβ receptor in some embodiments. In some embodiments, the IL-2 mutant protein of the present invention exhibits reduced binding affinity to the IL-2Rβγ receptor compared to the protein before attenuation, reduced by, for example, 1 to 100 times or more. In some embodiments, the IL-2 mutant protein of the present invention does not bind to the IL-2Rβ receptor but can still bind to the IL-2Rβγ receptor, preferably, the binding to the IL-2Rβγ receptor can be reduced by 1 to 100 times compared to the protein before attenuation, for example, by about 20 to 80 times. The ForteBio affinity measurement technology determines the equilibrium dissociation constant (K) of the IL-2 mutant protein of the present invention, such as the IL-2 mutant protein of the present invention fused with an Fc fragment or its dimer molecule, and the receptor IL-2Rβ or IL-2Rβγ receptor. D By measuring this, the binding affinity can be determined.

[0057] In some embodiments, by introducing a mutation at the IL-2Rβγ binding interface, the IL-2 mutant protein of the present invention has weakened IL-2 activity, such as IL-2 activity selected from at least one of the following, compared to the corresponding protein before the introduction of the mutation: - Compared to the protein before weakening, there is a reduction in activation of T cells (e.g., CD4+ and CD8+ T cells, e.g., CD4+ / CD8+CD25- T cells, CD4+CD25+ T cells). - Compared to the protein before weakening, the activation of NK cells was reduced. - Reduced release of inflammatory factors by IL-2-stimulated T cells / NK cells compared to the protein before weakening.

[0058] In one embodiment, compared to the protein before attenuation, the IL-2 mutant protein of the present invention results in reduced activation and / or proliferation of IL-2-mediated lymphocytes (e.g., T cells and / or NK cells). In one embodiment, lymphocytes are CD25 - These are CD4+ and CD8+ T cells, such as T cells. In one embodiment, the ability of the IL-2 mutant protein to activate CD4+ and CD8+ T cells in a STAT5 phosphorylation assay is identified by detecting the activation of the STAT5 phosphorylation signal in lymphocytes such as T cells or NK cells by the IL-2 mutant protein. For example, the maximum half-volume effective concentration (EC50) can be determined by analyzing STAT5 phosphorylation in cells by flow cytometry, as described in the examples of this application. For example, by detecting the ratio of the EC50 values ​​that activate the STAT5 phosphorylation signal in T cells of the IL-2 attenuated molecule of the present invention and the corresponding protein, the IL-2 mutant molecule of the present invention has "attenuated" T cell activation activity. Based on this ratio, the T cell activation activity of the IL-2 mutant molecule of the present invention can be reduced by, for example, 5 times or more, for example, 10 times or more, or 50 times or more, or 100 times or more, or even 1000 times or more. For example, compared to the corresponding protein, the T cell activation activity of the IL-2 mutant molecule of the present invention can be reduced by a factor of 10 to 50, 50 to 100, 100 to 1000, or more. In some preferred embodiments, the IL-2 mutant protein of the present invention has reduced cell surface IL-2 receptor-mediated IL-2 clearance and exhibits an increased in vivo half-life compared to wild-type IL-2.

[0059] In some preferred embodiments, the IL-2 mutant protein of the present invention has reduced IL-2 and receptor-mediated in vivo toxicity compared to wild-type IL-2.

[0060] Maintained or altered IL-2Rα receptor binding

[0061] The IL-2 protein initiates signal transduction and exerts its function by interacting with the IL-2 receptor. Wild-type IL-2 exhibits different affinities to different IL-2 receptors. IL-2β and γ receptors, which have low affinity for wild-type IL-2, are used in quiescent effector cells (CD8). + It is expressed on T cells and NK cells. IL-2Rα, which has a high affinity for wild-type IL-2, is 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, further recruiting IL-2Rβγ, which releases downstream p-STAT5 signaling via IL-2Rβγ, stimulating Treg cells and activated effector cells. Without theory, altering the affinity of IL-2 for the IL-2Rα receptor leads to CD25 + This alters the bias of IL-2 in preferentially activating cells, thereby changing the IL-2-mediated immune downregulation effect of Treg cells.

[0062] In some embodiments, the IL-2 mutant protein of the present invention has maintained or altered IL-2Rα receptor binding ability compared to wild-type IL-2.

[0063] In some embodiments, the IL-2 mutant protein of the present invention maintains binding to the IL-2Rα receptor compared to wild-type IL-2. In this specification, the expression "maintaining binding to the IL-2Rα receptor" means that the IL-2 mutant protein has equivalent binding activity to the IL-2Rα receptor compared to wild-type IL-2 protein. Preferably, "equivalent binding activity" means that, when measured by the same measurement method, the binding activity values ​​(e.g., binding affinity K) of the IL-2 mutant protein and wild-type IL-2 protein are equal. D The ratio between ) is between 1:20 and 20:1, preferably between 1:10 and 10:1. Preferably, the IL-2 mutant protein does not have mutations in the IL-2Rα binding interface compared to wild-type IL-2.

[0064] In some embodiments, the IL-2 mutant protein of the present invention is a weakened IL-2 mutant molecule that maintains binding to the IL-2Rα receptor. In some further embodiments, the weakened IL-2 mutant protein of the present invention does not have mutations at the IL-2Rα binding interface compared to wild-type IL-2. Preferably, the weakened IL-2 mutant molecule exhibits improved Treg selectivity and / or improved NK cell (e.g., CD3) - CD56 + It exhibits selectivity for NK cells. In one embodiment, in a STAT5 phosphorylation assay, the selectivity of the IL-2 mutant protein for lymphocytes is identified by detecting the activation of the STAT5 phosphorylation signal by the IL-2 mutant protein in different lymphocytes, such as Treg cells, NK cells, and CD4+ and CD8+ effector T cells. In one embodiment, in a STAT5 phosphorylation assay, the selectivity of the IL-2 mutant protein can be reflected by an IL-2 mutant protein dose window that selectively activates specific (one or more) lymphocytes without substantially activating other lymphocytes. For example, in some embodiments, the weakened IL-2 mutant protein of the present invention is CD25 - / low Compared to effector T cells such as CD4+ and / or CD8+ effector T lymphocytes, improved Treg selectivity and / or improved NK cell (CD3 - CD56 + NK cell selectivity can be demonstrated. In some further embodiments, the improved selectivity can be reflected by the lower drug-related toxicity of the IL-2 mutant protein.

[0065] In several other embodiments, the IL-2 mutant protein of the present invention introduces mutations in the IL-2Rα binding interface that reduce or eliminate IL-2Rα receptor binding compared to wild-type IL-2.

[0066] In some further embodiments, the IL-2 mutant protein of the present invention has a CD25 ratio compared to wild-type IL-2. +The bias of IL-2 in preferentially activating cells is reduced. In some further embodiments, the IL-2 mutant protein of the present invention reduces the IL-2-mediated immunodownregulating effect on Treg cells compared to wild-type IL-2.

[0067] In some other embodiments, the IL-2 mutant protein of the present invention has an immunodownregulating effect. In some further embodiments, the IL-2 mutant protein of the present invention can be used to treat autoimmune diseases.

[0068] Therefore, in some embodiments, the IL-2 mutant protein of the present invention has improved properties selected from, for example, one or more of the following: - Compared to wild-type IL-2, maintenance or alteration (e.g., reduction or increase) of binding affinity to the high-affinity IL-2R receptor (IL-2Rαβγ), - Compared to wild-type IL-2, maintenance or alteration (e.g., reduction or increase) of activation in CD25+ cells (e.g., CD8+ T cells and Treg cells), - Maintenance or alteration of the bias of IL-2 to preferentially activate CD25+ cells (e.g., Treg cells) compared to wild-type IL-2 (e.g., removal, reduction, or increase). - Maintenance or alteration (e.g., reduction or increase) of the downregulation effect of IL-2-induced immune responses by Treg cells compared to wild-type IL-2.

[0069] In some embodiments, wild-type IL-2 (for example, IL-2 shown in Sequence ID No. 1) WTCompared to the original IL-2 mutant protein, the IL-2 mutant protein of the present invention exhibits a binding affinity to the IL-2Rα receptor that is reduced by at least 5 times, at least 10 times, or at least 25 times, and particularly at least 30 times, 50 times, or 100 times or more. In a preferred embodiment, the mutant protein of the present invention does not bind to the IL-2 receptor α. The equilibrium dissociation constant (K) of the IL-2 mutant protein of the present invention, such as the IL-2 mutant protein of the present invention fused with an Fc fragment or its dimer molecule, and the receptor IL-2Rα receptor is determined by ForteBio affinity measurement technology. D By measuring this, the binding affinity can be determined.

[0070] In one embodiment, compared to wild-type IL-2, the IL-2 mutant protein of the present invention is IL-2-mediated CD25 + This results in decreased cell activation and / or proliferation. In one embodiment, CD25 + Cells are CD25 + CD8 + In another embodiment, CD25 + The cells are Treg cells. In one embodiment, in a STAT5 phosphorylation measurement test, CD25 + The ability of IL-2 mutant proteins to activate cells is due to CD25 by IL-2 mutant proteins. + It is identified by detecting the activation of the STAT5 phosphorylation signal in cells. For example, the maximum effective concentration (EC50) can be determined by analyzing STAT5 phosphorylation in cells by flow cytometry, as described in the examples of this application.

[0071] In one embodiment, compared to wild-type IL-2, the IL-2 mutant protein of the present invention has CD25 + Eliminate or reduce the bias of IL-2 that preferentially activates cells. In one embodiment, CD25 + Cells are CD25 + CD8 + In another embodiment, CD25 +The cells are Treg cells. In one embodiment, in a STAT5 phosphorylation measurement test, CD25 - The ability of IL-2 mutants to activate cells is determined by their respective CD25 - Cells and CD25 + These are identified by detecting the EC50 value of IL-2 mutant proteins that activate the STAT5 phosphorylation signal in cells. For example, CD25 + The activation bias of IL-2 mutant proteins in cells is CD25 - and CD25 + This is determined by calculating the ratio of EC50 values ​​that activate the STAT5 phosphorylation signal in T cells. Preferably, CD25 compared to wild-type protein. + The bias of the mutant protein toward was reduced by at least 10 times, preferably at least 100 times, 150 times, 200 times, 300 times or more.

[0072] Mutant protein of the present invention

[0073] In one embodiment, the present invention provides an Il-2 mutant protein comprising the following mutations compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 containing sequence number 1): (i) A mutation at the binding interface of IL-2 to IL-2Rα, particularly at at least one position selected from positions 35, 37, 38, 41, 42, 43, 45, 61, 68 and 72, that eliminates or reduces the binding affinity to the IL-2Rα receptor, and / or (ii) A shortened B'C' loop region (i.e., a sequence linking amino acid residues aa72 and aa84), preferably having an amino acid length of 10, 9, 8, 7, 6, or less than 5, and preferably having an amino acid length of 7, wherein the shortened B'C' loop region results in an improvement in protein expression level and / or purity, And including the following mutations: (iii) A mutation at the binding interface of IL-2 to IL-2Rβγ, particularly at at least one position selected from positions 12, 15, 16, 19, 20, 84, 87, 88, 91, 92, 95, and 126, which weakens the binding to the IL-2Rβγ receptor. Here, the amino acid positions are numbered according to Sequence ID No. 1. Preferably, the mutant protein comprises mutations (i) and (iii), or mutations (ii) and (iii), or mutations (i), (ii) and (iii).

[0074] Mutation of the IL-2Rβγ binding interface

[0075] The mutations of the IL-2Rβγ binding interface applied to the mutant protein of the present invention may be any mutations that, when combined with other mutations of the present invention, result in weakening of IL-2Rβγ binding affinity and / or weakening of activity that activates lymphocytes (e.g., T cells / NK cells).

[0076] Examples of such mutations include, but are not limited to, mutations at the IL-2 binding interface with IL-2Rβγ, particularly at at least one position selected from positions 12, 15, 16, 19, 20, 84, 87, 88, 91, 92, 95, and 126, that result in weakened IL-2Rβγ receptor binding.

[0077] In some embodiments, mutations in the IL-2Rβγ binding interface are L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16T, H16Y, H16A, H 16E, H16D, H16R, L19D, L19E, L19R, L19S, D20N, D20Q, D20E, D20A, D20R, D2 0S, D84N, D84E, D84Q, D84T, D84S, D84R, D84G, D84M, D84F, D84L, D84K, D84 H, S87T, S87R, S87K, S87L, S87M, S87H, N88D, N88T, N88Q, N88R, N88E, N88K This includes mutations selected from N88H, N88M, N88S, N88L, V91I, V91L, V91D, V91E, V91N, V91Q, V91S, V91H, I92E, I92T, I92K, I92R, I92L, E95Q, E95G, E95D, E95N, Q126E, Q126D, Q126A, Q126S, D84N+E95Q, D84E+E95Q, D84T+E95Q, D84Q+E95Q, D84T+H16T, D84N+V91I, D84T+Q126E, D84N+Q126E, H16T+D84Q, and H16T+V91I.

[0078] In some preferred embodiments, the mutation at the IL-2Rβγ binding interface is Includes mutations selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88R, N88D, N88Q, N88T, Q126E, and V91I.

[0079] In some further preferred embodiments, the mutation of the IL-2Rβγ binding interface is - A mutation selected from D84N, E95Q, H16N, or - A mutation selected from D84Q, H16T+V91I, or - A mutation selected from D84T, Q126E, D84N+V91I, and D84N+E95Q, or - A mutation selected from N88D, N88R, D20N, D84N+E95Q, D84T+H16T, D84T+Q126E, D84N+Q126E, and H16T+D84Q, or In some further preferred embodiments, the mutations of the IL-2Rβγ binding interface include mutations selected from D20N, N88D, and N88R.

[0080] B'C' loop region mutation

[0081] In one embodiment, the IL-2 mutant protein of the present invention comprises a mutation in the B'C' loop region compared to wild-type IL-2, preferably the mutation resulting in improved stability of the B'C' loop region, and more preferably the mutation resulting in the IL-2 mutant protein of the present invention having improved drug potential, such as increased expression level and / or purity.

[0082] In some embodiments, the introduced mutation results in the mutant protein containing a shortened B'C' loop region (i.e., a shortened ligation sequence length between amino acid residues aa72 and aa84) compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 containing the SEQ ID NO: 1 sequence).

[0083] Preferably, the shortened loop region has an amino acid length of 10, 9, 8, 7, 6, or less than 5, and preferably 7 amino acid lengths, where the amino acid residues are numbered according to Sequence ID No. 1.

[0084] In this specification, mutations in the B'C' loop region applicable to the present invention include cleavage and substitution of the B'C' loop region. In one embodiment, the mutation includes cleavage or substitution of amino acid residues aa73 to aa83 in the B'C' loop region, such as cleavage to form A(Q / G)SKN(F / I)H or substitution by SGDASIH. In other embodiments, the mutation includes cleavage or substitution of amino acid residues aa74 to aa83 in the B'C' loop region, such as cleavage to form (Q / G)SKN(F / I)H or substitution by GDASIH.

[0085] In some embodiments, the IL-2 mutant protein of the present invention comprises a B'C' loop chimeric mutation. Compared to wild-type IL-2, the mutant protein comprises substitutions to all or part of the sequence linking aa72-aa84, such as substitutions with a short B'C' loop sequence from a member of another 4-helix short-chain cytokine family. Short B'C' loops applicable to substitute wild-type IL-2 can be identified by superpose of the crystal structure from other members of the 4-helix short-chain cytokine IL family, such as IL-15, IL-4, IL-21, or from members of the IL family derived from non-human species (e.g., mouse). In one embodiment, the sequence for substitution is a B'C' loop sequence derived from interleukin IL-15 (particularly human IL-15). In one embodiment, the substitution comprises substitutions of amino acid residues aa73-aa83 in the B'C' loop region. In other embodiments, the substitution comprises substitutions of amino acid residues aa74-aa83 in the B'C' loop region. Preferably, after substitution, the IL-2 mutant protein of the present invention has a B'C' loop sequence selected from SGDASIH or AGDASIH (i.e., a sequence linking aa72 to aa84).

[0086] In some embodiments, the IL-2 mutant protein of the present invention comprises a B'C' loop cleavage mutation. Compared to wild-type IL-2, the mutant protein comprises a cleavage of a sequence linking aa72-aa84. In one embodiment, the cleavage comprises a cleavage of amino acid residues aa73-aa83 in the B'C' loop region. In other embodiments, the cleavage comprises a cleavage of amino acid residues aa74-aa83 in the B'C' loop region. For example, one, two, three, or four amino acids can be cleaved from the C-terminus. Preferably, after cleavage, the B'C' loop region of the IL-2 mutant protein of the present invention has the sequence A(Q / G)SKN(F / I)H. Preferably, after cleavage, the IL-2 mutant protein of the present invention has a B'C' loop sequence (i.e., a sequence linking aa72-aa84) selected from the following:

[0087] [Table 2]

[0088] In one preferred embodiment, the IL-2 mutant protein of the present invention comprises a B'C' loop region sequence selected from AQSKNFH, SGDASIH, or AGDASIH (i.e., a sequence ligating aa72 to aa84).

[0089] For the B'C' loop mutation applicable to the present invention, one can also refer to the applicant's concurrently pending application PCT / CN2019 / 107054, which is incorporated herein by reference in its entirety.

[0090] Mutation of the IL-2Rα binding interface

[0091] In one embodiment, the IL-2 mutant protein of the present invention contains one or more mutations at the IL-2Rα binding interface, preferably at positions 35, 37, 38, 41, 42, 43, 45, 61, 68, and 72, compared to wild-type IL-2. Preferably, the mutations eliminate or reduce the binding affinity to the IL-2Rα receptor.

[0092] In some embodiments, the mutations of the IL-2Rα binding interface of the present invention include a combination of mutations selected from one of the following combinations (1) to (9):

[0093] [Table 3]

[0094] In some preferred embodiments, the mutations of the IL-2Rα binding interface of the present invention include or consist of the mutation combination K35E+T37E+R38E+F42A.

[0095] In some other preferred embodiments, the mutations of the IL-2Rα binding interface of the present invention include or consist of the mutation combination K35E+T37E+R38E.

[0096] In some other preferred embodiments, the mutation of the IL-2Rα binding interface of the present invention includes or consists of mutation F42A.

[0097] In several other embodiments, the IL-2 mutant protein of the present invention, which includes a mutation in the IL-2Rα binding interface of the present invention, has altered IL-2Rα binding, such as altered IL-2Rα binding as measured by ForteBio affinity assay.

[0098] For variations of the IL-2Rα binding interface applicable to the present invention, one can also refer to the applicant's concurrently pending application PCT / CN2019 / 107055, which is incorporated herein by reference in its entirety.

[0099] Preferred exemplary mutation combinations

[0100] In some preferred embodiments, the IL-2 mutant protein of the present invention has a weakened IL-2Rβγ bond and has improved properties selected from one or all of the following: (i) reduced (or eliminated) IL-2Rα bond, and (ii) improved expression level and purity.

[0101] In some embodiments, the present invention, compared to wild-type IL-2, (a) Mutation combination K35E+T37E+R38E+F42A, (b) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, provides IL-2 mutant proteins including (iv) mutant T3A.

[0102] In some embodiments, the present invention, compared to wild-type IL-2, (a) Mutation combination K35E+T37E+R38E, (b) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, provides IL-2 mutant proteins including (iv) mutant T3A.

[0103] In some embodiments, the present invention, compared to wild-type IL-2, (a) Mutation F42A, (b) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, provides IL-2 mutant proteins including (iv) mutant T3A.

[0104] In some embodiments, the present invention, compared to wild-type IL-2, (a) B'C' loop region array AQSKNFH, (b) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, The present invention also provides an IL-2 mutant protein containing, optionally, (iv) mutant T3A. Preferably, the IL-2 mutant protein maintains IL-2Rα receptor binding ability compared to the wild-type IL-2 protein.

[0105] In some embodiments, the present invention, compared to wild-type IL-2, (a) B'C' loop region sequence SGDASIH or AGDASIH, (b) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, The present invention also provides an IL-2 mutant protein containing, optionally, (iv) mutant T3A. Preferably, the IL-2 mutant protein maintains IL-2Rα receptor binding compared to the wild-type IL-2 protein.

[0106] In some embodiments, the IL-2 mutant protein of the present invention is compared to wild-type IL-2, (i) Mutation combination K35E+T37E+R38E+F42A, or mutation combination K35E+T37E+R38E, or mutation F42A, (ii) B'C' loop region sequence AQSKNFH or SGDASIH or AGDASIH, (iii) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, including (iv) mutation T3A.

[0107] In some embodiments, the IL-2 mutant protein of the present invention is compared to wild-type IL-2, (i) Mutation combination K35E+T37E+R38E+F42A, (ii) B'C' loop region sequence AQSKNFH, (iii) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, including (iv) mutation T3A.

[0108] In some embodiments, the IL-2 mutant protein of the present invention is compared to wild-type IL-2, (i) Mutation combination K35E+T37E+R38E+F42A, (ii) B'C' loop region sequence SGDASIH or AGDASIH, (iii) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, including (iv) mutation T3A.

[0109] In some embodiments, the IL-2 mutant protein of the present invention is compared to wild-type IL-2, (i) Mutation combination K35E+T37E+R38E, (ii) B'C' loop region sequence AQSKNFH, (iii) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, including (iv) mutation T3A.

[0110] In some embodiments, the IL-2 mutant protein of the present invention is compared to wild-type IL-2, (i) Mutation combination K35E+T37E+R38E, (ii) B'C' loop region sequence SGDASIH or AGDASIH, (iii) Variations of the IL-2Rβγ binding interface selected from D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I, And, in some cases, including (iv) mutation T3A.

[0111] In some preferred embodiments, the IL-2 mutant protein of the present invention, compared to wild-type IL-2, (i) Mutation combination K35E+T37E+R38E+F42A, (ii) B'C' loop region sequence SGDASIH or AGDASIH, (iii) Variation of the IL-2Rβγ binding interface D84N or D84N+E95Q, And, optionally, (iv) mutation T3A. More preferably, the IL-2 mutant protein of the present invention comprises, compared to wild-type IL-2, (i) the combination of mutations K35E+T37E+R38E+F42A, (ii) the B'C' loop region sequence AGDASIH, and (iii) the mutation D84N+E95Q at the IL-2Rβγ binding interface.

[0112] In some further preferred embodiments, the IL-2 mutant protein of the present invention maintains binding to the IL-2Rα receptor compared to the wild-type IL-2 protein, and (i) B'C' loop region array AQSKNFH, SGDASIH, or AGDASIH, (ii) Variations of the IL-2Rβγ binding interface selected from D20N, N88R, and N88D, And, optionally, (iii) mutant T3A. Preferably, the IL-2 mutant protein does not have mutations at the IL-2Rα binding interface compared to wild-type IL-2.

[0113] In several other preferred embodiments, the IL-2 mutant protein of the present invention maintains binding to the IL-2Rα receptor compared to the wild-type IL-2 protein, and (i) B'C' loop region array SGDASIH or AGDASIH, (ii) Variations of the IL-2Rβγ binding interface: D20N, N88R, or N88D, And, optionally, (iii) mutant T3A. Preferably, the IL-2 mutant protein does not have mutations at the IL-2Rα binding interface compared to wild-type IL-2.

[0114] In several other preferred embodiments, the IL-2 mutant protein of the present invention maintains binding to the IL-2Rα receptor compared to the wild-type IL-2 protein, and (i) B'C' loop region array AGDASIH, (ii) Variations of the IL-2Rβγ binding interface: D20N, N88R, or N88D, And, optionally, (iii) mutant T3A. Preferably, the IL-2 mutant protein does not have mutations at the IL-2Rα binding interface compared to wild-type IL-2.

[0115] In some further preferred embodiments, the IL-2 mutant protein of the present invention maintains binding to the IL-2Rα receptor compared to wild-type IL-2 (e.g., SEQ ID NO: 1), and (i) B'C' loop region array AGDASIH, (ii) Mutation N88R at the IL-2Rβγ bond interface, and (iii) mutant T3A. Preferably, the IL-2 mutant protein does not have mutations at the IL-2Rα binding interface compared to wild-type IL-2. In one embodiment, the IL-2 mutant protein of the present invention has a maturation region that has at least 85% or 90% identity in its amino acid sequence with the maturation region of the wild-type IL-2 protein listed in one of SEQ ID NOs: 1-3.

[0116] In some preferred embodiments, the IL-2 mutant protein of the present invention comprises an amino acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, 99% identity with an amino acid sequence selected from one of SEQ ID NOs: 37-638 (particularly SEQ ID NOs: 469-553 or 554-638, preferably SEQ ID NOs: 148 or 197, more preferably SEQ ID NOs: 489, 513, 516). In some embodiments, the mutant protein comprises or consists of an amino acid sequence selected from SEQ ID NOs: 37-638. In some preferred embodiments, the mutant protein comprises or consists of an amino acid sequence selected from SEQ ID NOs: 469-553 or 554-638, preferably SEQ ID NOs: 148 or 197, more preferably SEQ ID NOs: 489, 513, 516.

[0117] Other mutations

[0118] Apart from the above-mentioned "IL-2Rβγ binding interface mutations," "B'C' loop region mutations," and "IL-2Rα binding interface mutations," the IL-2 mutant protein of the present invention may further have one or more mutations in other regions or positions, provided that it retains one or more of the above-mentioned beneficial properties of the IL-2 mutant protein of the present invention. For example, the IL-2 mutant protein of the present invention may further include substitutions at position 125, e.g., C125S, C125A, C125T, or C125V, to provide further advantages such as improved expression, homogeneity, or stability (see, for example, U.S. Patent No. 4,518,584). Furthermore, for example, the IL-2 mutant protein of the present invention may further include substitutions at position 3, such as T3A, to remove the O-sugar modification at the N-terminus of IL2. Those skilled in the art will know how to determine additional mutations that can be incorporated into the IL-2 mutant protein of the present invention.

[0119] Sequence differences between the IL-2 mutant protein and the wild-type protein can be represented by sequence identity or by the number of different amino acids between them. In one embodiment, the IL-2 mutant protein and the wild-type protein have at least 85%, 86%, 87%, 88%, or 89% identity, preferably 90% or more, preferably 95%, but preferably 97% or less, and more preferably 96% or less identity. In another embodiment, apart from the three types of mutations described above, the IL-2 mutant protein and the wild-type protein may have 15 or fewer mutations, for example, 1 to 10, or 1 to 5, for example, 0, 1, 2, 3, or 4 mutations. In one embodiment, the remaining mutations may be conservative substitutions.

[0120] 2. Fusion proteins and IL-2-Fc dimer proteins

[0121] In one embodiment, the present invention further provides a fusion protein comprising the IL-2 mutant protein of the present invention. In one preferred embodiment, the IL-2 mutant protein of the present invention is fused with another polypeptide, such as albumin, more preferably an antibody Fc fragment, which can confer improved pharmacokinetic properties.

[0122] In one embodiment, the present invention provides an IL-2 mutant protein fusion protein comprising the IL-2 mutant protein of the present invention fused with an antibody Fc fragment.

[0123] The Fc fragments used in the present invention may contain mutations that reduce or eliminate effector function. In one preferred embodiment, the Fc fragment has effector function mediated by a reduced Fc region, for example, reduced or eliminated ADCC, ADCP, or CDC effector function. For example, in some special embodiments, the Fc fragments used in the present invention have an L234A / L235A mutation or L234A / L235E / G237A that reduces binding to the Fcγ receptor.

[0124] In a further preferred embodiment, the Fc fragment may have a mutation that increases its serum half-life, for example, a mutation that improves the binding of the Fc fragment to FcRn.

[0125] In some embodiments, the Fc fragment fused with the IL-2 mutant protein is a human IgG Fc such as human IgG1 Fc, human IgG2 Fc, or human IgG4 Fc. In one embodiment, the Fc fragment contains amino acid sequence number 12 or has at least 90% identity with it, for example, 95%, 96%, 97%, 99%, or more.

[0126] In some embodiments, the IL-2 mutant protein is fused to the Fc region by a linker. In some embodiments, CD25 - To enhance the activation effect of Fc fusion proteins on T cells, a linker can be selected. In one embodiment, the linker is GSGS, more preferably (G4S)2.

[0127] In a further embodiment, the present invention also provides a dimerized molecule comprising the IL-2 mutant protein of the present invention fused with an Fc fragment. Such a dimerized molecule can increase the molecular weight to 60-80 kDa, significantly reduce renal clearance, and further extend the half-life of the IL2-Fc fusion protein through in vivo FcRn-mediated recycling. Preferably, the dimerized molecule has one or more of the following properties compared to a corresponding dimerized molecule comprising the wild-type IL-2-Fc fusion protein: - Increased expression level and / or purity when expressed in mammalian cells (e.g., CHO or HEK293 cells), - Reduction or avoidance of excessive lymphocyte activation and / or release of inflammatory factors by IL-2, - For example, better pharmacokinetic properties such as a delayed in vivo half-life, - Low toxicity when administered in vivo, such as low cardiovascular toxicity (e.g., low vascular leakage side effects).

[0128] Preferably, the dimeric molecule of the IL-2 mutant protein of the present invention exhibits good antitumor effects and resistance when administered to animals. The antitumor effect can be determined by measuring the tumor suppression rate in the tumor-bearing animal experiments described in the examples. Resistance can be determined by detecting the body weight of the animal model after administration and the changes in body weight, as described in the examples.

[0129] In some embodiments, the IL-2-Fc dimer protein provided by the present invention is a homodimer in which the first and second monomers comprise, from the N-terminus to the C-terminus, i) an IL-2 mutant protein, ii) a linker, and iii) an Fc fragment.

[0130] In some other embodiments, the IL-2-Fc dimer protein provided by the present invention is a heterodimer, a) From the N-terminus to the C-terminus, i) an IL-2 mutant protein, ii) a linker, and iii) a first monomer containing a first Fc fragment, b) comprising a second monomer containing a second Fc fragment.

[0131] In some embodiments, the first Fc fragment and the second Fc fragment each contain first and second heterodimerizing mutations that promote the formation of a heterodimer between the first monomer and the second monomer. In some preferred embodiments, the first and second heterodimerizing mutations include combinations of Knob:Hole mutations, such as the mutation combination T366W / S354C:Y349C / T366S / L368A / Y407V.

[0132] In some preferred embodiments, the first heterodimer mutation in the first Fc fragment comprises a Knob mutation and the second heterodimer mutation in the second Fc fragment comprises a Hole mutation, or the first heterodimer mutation in the first Fc fragment comprises a Hole mutation and the second heterodimer mutation in the second Fc fragment comprises a Knob mutation.

[0133] As those skilled in the art will understand, the Fc fragment of the fusion protein and dimer molecule applied to the present invention may be any antibody Fc fragment. In one embodiment, the Fc fragment of the present invention is effector-silent.

[0134] In one embodiment, an Fc fragment is modified with one or more properties selected from effector function and complement activation function of the Fc region. In one embodiment, the effector function or complement activation function is reduced or eliminated compared to the wild-type Fc region of the same isotype. In one embodiment, the effector function is reduced or eliminated by methods such as reducing glycosylation of the Fc region, using an Fc isotype that naturally has the reduced or eliminated effector function, and modifying the Fc region.

[0135] In one embodiment, effector function is reduced or eliminated by reducing glycosylation of the Fc region. In one embodiment, glycosylation of the Fc region is reduced by methods such as producing the fusion protein or dimer molecule of the present invention in an environment that does not tolerate wild-type glycosylation, removing a carbohydrate group already present in the Fc region, and modifying the Fc region so that wild-type glycosylation does not occur. In one embodiment, glycosylation of the Fc region is reduced by a method of modification so that wild-type glycosylation does not occur, and includes, for example, a mutation at position 297 of the Fc region, such as the N297A mutation, so that the wild-type asparagine residue at the following position is replaced with another amino acid that interferes with glycosylation at that position.

[0136] In one embodiment, the effector function is reduced or eliminated by modifying at least one Fc region. In one embodiment, the modification of at least one Fc region is at positions 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 3 The modification is selected from point mutations in the Fc region that disrupt binding to one or more Fc receptors, selected from 82, 388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; point mutations in the Fc region that disrupt binding to C1q, selected from positions 270, 322, 329, and 321; and a point mutation at position 132 of the CH1 structural domain. In one embodiment, the modification is a point mutation in the Fc region selected from positions 270, 322, 329, and 321 that disrupts binding to C1q. In another embodiment, the modification eliminates several Fc regions.

[0137] As will be understood by those skilled in the art, in order to facilitate the formation of the heterodimer of the present invention, the Fc fragment of the dimer molecule of the present invention may contain mutations favorable to the dimerization of the first monomer and the second monomer. Preferably, based on the Knob-in-Hole technique, knob mutations and hole mutations corresponding to the first monomer and the second monomer are introduced.

[0138] Therefore, in one embodiment, the dimer molecule of the present invention comprises the following: i) In some cases, the homodimer Fc-region of a human IgG1 subclass having mutations P329G, L234A and L235A, or ii) In some cases, the Fc-region of a human IgG4 subclass homodimer having mutations P329G, S228P, and L235E, or iii) A heterodimer Fc-region, of which a) One Fc-region polypeptide contains mutation T366W, another Fc-region polypeptide contains mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains mutations T366W and Y349C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and S354C, or c) Heterodimeric Fc-region polypeptides, one containing mutants T366W and S354C, and another containing mutants T366S, L368A, Y407V and Y349C, or iv) A heterodimer Fc-region of a human IgG4 subclass, wherein both of the Fc-region polypeptides contain mutants P329G, L234A, and L235A, and a) One Fc-region polypeptide contains mutation T366W, another Fc-region polypeptide contains mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains mutations T366W and Y349C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and S354C, or c) A heterodimer Fc region of a human IgG4 subclass, in which one Fc-region polypeptide contains mutations T366W and S354C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and Y349C. or v) A heterodimer Fc-region of a human IgG4 subclass, wherein both of the Fc-region polypeptides contain mutants P329G, S228P, and L235E, and a) One Fc-region polypeptide contains mutation T366W, another Fc-region polypeptide contains mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains mutations T366W and Y349C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and S354C, or c) A heterodimer Fc-region of a human IgG4 subclass, in which one Fc-region polypeptide contains mutations T366W and S354C, and the other Fc-region polypeptide contains mutations T366S, L368A, Y407V and Y349C.

[0139] In some embodiments, the Fc region may further include other mutations advantageous for the purification of the heterodimer. For example, to facilitate the purification of the heterodimer using protein A, the H435R mutation (Eric J. Smith, Scientific Reports 5:17943 DOI:10.1038 / srep17943) may be introduced into one of the Fc regions of the heterodimer (e.g., the Fc region with the Hole mutation). In some other embodiments, with respect to heterodimer monomers including a hinge region, mutations such as C220S may also be introduced into the hinge region to facilitate the formation of the heterodimer.

[0140] As will be apparent to those skilled in the art, the linker connecting the IL-2 mutant protein and the Fc fragment in the fusion protein and dimer molecule to which the present invention applies may be any linker known in the art. In some embodiments, the linker may include an IgG1 hinge, or it may include a linker sequence selected from (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least an integer of 1. Preferably, the linker includes (G4S)2, i.e., GGGGSGGGGS.

[0141] Accordingly, in one preferred embodiment, each monomer in the dimer of the present invention comprises an IL-2 mutant protein selected from SEQ ID NOs. 37-638 (particularly SEQ ID NOs. 469-553 or 554-638, preferably SEQ ID NOs. 489, 513, 516) linked at the C-terminus to the SEQ ID NO. 12 amino acid sequence via linker (G4S) 2. In some preferred embodiments, the first monomer of the dimer molecule comprises an IL-2 mutant protein selected from SEQ ID NOs. 37-638 (particularly SEQ ID NOs. 469-553 or 554-638, preferably SEQ ID NOs. 489, 513, 516) linked at the C-terminus to the SEQ ID NO. 9 amino acid sequence via linker (G4S) 2, and the second monomer comprises the amino acid sequence of SEQ ID NO. 10.

[0142] 3. Immune complex

[0143] The present invention further provides an immune complex comprising the IL2 mutant protein and antigen-binding molecule of the present invention. Preferably, the antigen-binding molecule is an immunoglobulin molecule, particularly an IgG molecule, or an antibody or antibody fragment, particularly Fab molecules and scFv molecules. In some embodiments, the antigen-binding molecule specifically binds to antigens present on tumor cells or in the tumor environment, such as antigens selected from fibroblast-activating protein (FAP), the A1 domain of tenascin C (TNC A1), the A2 domain of tenascin C (TNC A2), the extra domain B (EDB) of fibronectin, carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP). Thus, the immune complex 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 lower doses and thereby reduced side effects, and enhanced antitumor effects.

[0144] In the immune complex of the present invention, the IL-2 mutant protein of the present invention can be bound directly or via a linker to another molecule or antigen-binding molecule, and in some embodiments, a protein hydrolysis cleavage site is included between them.

[0145] 4. Polynucleotides, vectors, and hosts

[0146] The present invention provides nucleic acids encoding any of the aforementioned IL-2 mutant proteins, fusion proteins, dimeric molecules, or complexes. The polynucleotide sequences encoding the mutant proteins of the present invention can be produced by novel solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding wild-type IL-2, using methods well known in the art. The polynucleotides and nucleic acids of the present invention may also include segments encoding secretory signal peptides, which can be operably linked to segments encoding the mutant proteins of the present invention to induce secretory expression of the mutant proteins of the present invention.

[0147] The present invention also provides vectors comprising nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes, but is not limited to, a virus, plasmid, cosmid, lambda phage, or yeast artificial chromosome (YAC). In a preferred embodiment, the expression vector of the present invention is a pYDO_017 expression vector.

[0148] The present invention also provides host cells containing the nucleic acid or the vector. Host cells applied to replicate and support the expression of mutant IL-2 proteins or fusions or dimers or immune complexes are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and many of these vector-containing cells can be grown to inoculate large fermenters, yielding sufficient quantities of IL-2 mutants or fusions or dimers or immune complexes for clinical use. In one embodiment, the host cells are eukaryotic cells. In another embodiment, the host cells are selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human fetal kidney line (e.g., 293 or 293T cells described in Graham et al., JGenVirol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, BiolReprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), and TRI cells (e.g., Mather et al.). These include MRC5 cells and FS4 cells (as described in al., Annals N.Y. AcadSci 383, 44-68 (1982)). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., ProcNatl AcadSci USA 77, 4216 (1980)), and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells).

[0149] 5. Preparation method

[0150] In a further embodiment, the present invention provides a method for preparing the IL-2 mutant protein or fusion or dimer or complex of the present invention, the method comprising the steps of culturing host cells, such as those provided above, containing nucleic acids encoding the protein or fusion or dimer or complex, under conditions suitable for the expression of the IL-2 mutant protein or fusion or dimer or complex, and optionally recovering the protein or fusion or dimer or complex from the host cells (or host cell medium).

[0151] 6.Measurement method

[0152] Various measurement methods known in the field can be used to identify, screen, or characterize the physical / chemical properties and / or biological activity of the IL-2 mutant proteins provided herein.

[0153] In one embodiment, the binding activity of the IL-2 mutant protein of the present invention to the IL-2 receptor can be measured. For example, the binding to human IL-2Rα or β protein or IL-2Rβγ or IL-2Rαβγ can be measured by methods known in the art, such as ELISA or Western blotting, or by exemplary methods disclosed in the examples herein. For example, it can be measured by flow cytometry in which cells such as yeast display cells transfected to express the mutant protein on the cell surface are reacted with labeled (e.g., biotin-labeled) IL-2Rα or β protein or IL-2Rβγ or IL-2Rαβγ complexes. Alternatively, binding dynamics (e.g., K) can be measured. D The binding of mutant proteins to receptors, including their values, can be measured using the ForteBio assay method with the IL-2-Fc fusion or dimeric molecular form.

[0154] In a further embodiment, the ability of IL-2 mutant proteins to bind to the IL-2 receptor can be indirectly measured by measuring the signal transduction and / or immunostimulatory effects occurring downstream of receptor binding.

[0155] Accordingly, in some embodiments, the present invention provides methods for identifying mutant IL-2 proteins having biological activity. Biological activity may include, for example, the ability to induce proliferation of IL-2 receptor-possessing T and / or NK cells and / or Treg cells, the ability to induce IL-2 signaling in IL-2 receptor-possessing T and / or NK cells and / or Treg cells, the ability to induce apoptosis in reduced T cells, the ability to induce tumor regression and / or improve 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, their Fc fusions and dimer molecules containing the same.

[0156] Methods well known in the field can be used to measure the biological activity of IL-2. For example, a suitable assay for testing the ability of the present invention's mutant IL-2 protein to stimulate IFN-γ production by NK cells (e.g., in the form of a dimeric molecule) may include the steps of incubating cultured NK cells with the mutant IL-2 protein of the present invention, and then measuring the IFN-γ concentration in the culture medium by ELISA. IL-2 signaling induces several signaling pathways and involves JAK (Janus kinase) and STAT (signaling molecule and transcriptional activator) signaling molecules.

[0157] The interaction between IL-2 and the receptor's β and γ subunits results in the phosphorylation of the receptor and JAK1 and JAK3 (which bind to the β and γ subunits, respectively). Subsequently, STAT5 binds to the phosphorylated receptor and is itself phosphorylated on a crucial tyrosine residue. This causes STAT5 to dissociate from the receptor, dimerize, and translocate the STAT5 dimer to the cell nucleus, where it promotes the transcription of the target gene. Therefore, the ability of a mutant IL-2 polypeptide to induce signal transduction via the IL-2 receptor can be evaluated, 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 polypeptide or fusion, dimer, or immune complex of the present invention, and the level of phosphorylated STAT5 can be measured by flow cytometry.

[0158] Furthermore, the effects of mutant IL-2 on tumor growth and survival can be evaluated in various animal tumor models known in the field. For example, xenografts of cancer cell lines can be transplanted into immunodeficient mice and treated with the mutant IL-2 polypeptide, fusion, dimer, or immune complex of the present invention. The in vivo antitumor effect of the mutant IL-2 polypeptide, fusion, dimer, and immune complex of the present invention can be detected based on the tumor suppression rate (e.g., calculated in comparison to isotype control antibodies). In addition, the in vivo toxicity of the mutant IL-2 polypeptide, fusion, dimer molecule, and immune complex of the present invention can be measured based on changes in the animal's body weight (e.g., absolute body weight change or percentage change in body weight compared to before administration). The in vivo toxicity can also be measured based on mortality, survival observation (visible symptoms of adverse effects such as behavior, body weight, and body temperature) and clinical and anatomical pathology (e.g., measurement of blood chemistry values ​​and / or histopathological analysis).

[0159] In a further embodiment, the drug potential of the mutant protein of the present invention, such as expression levels and product purity, can be characterized by methods known in the art. Regarding the measurement of expression levels, if the mutant protein is secreted from cultured cells and expressed in the culture supernatant, the protein content of the cell culture medium collected by centrifugation can be measured. Alternatively, it can be measured after a one-step purification of the collected cell culture medium, for example, after one-step affinity chromatography purification. Regarding the measurement of product purity, the purity of the mutant protein can be detected by measuring the purity after performing one-step affinity chromatography purification on the culture supernatant of the obtained production cells. Preferably, the mutant protein of the present invention has significantly better purity than the wild-type protein after purification by this one-step affinity chromatography, indicating that the mutant protein of the present invention has better purification performance. The purity measurement method includes, but is not limited to, the SEC-HPLC method, and may be any conventional method known in the art.

[0160] In a further embodiment, the pharmacokinetic properties of the mutant proteins, fusions, and dimer molecules of the present invention, such as their half-lives, can be characterized by methods known in the art.

[0161] 7. Method for modifying the IL-2 protein

[0162] In one embodiment, the present invention provides a method for obtaining an IL-2 mutant protein having improved properties and an IL-2 mutant protein obtained by this method.

[0163] In one embodiment, the method of the present invention is (1) The steps of shortening the loop region sequence of IL-2 by mutation in the B'C' loop region, and optionally introducing one or more mutations at the binding interface of IL-2 to IL-2Rβγ and / or introducing one or more mutations at the binding interface of IL-2 to IL-2Ra, (2) The step of expressing the IL-2 mutant protein in mammalian cells (e.g., HEK293 or CHO cells) in the form of, for example, an Fc fusion (e.g., an FcLALA fusion).

[0164] In a further embodiment, the method of the present invention is (1) Introducing one or more mutations at the binding interface of IL-2 to IL-2Rβγ, and optionally shortening the B'C' loop region sequence of IL-2 by mutation, and / or introducing one or more mutations at the binding interface of IL-2 to IL-2Ra, preferably in one embodiment not introducing a mutation at the binding interface of IL-2 to IL-2Ra, (2) The step of expressing the IL-2 mutant protein in mammalian cells (e.g., HEK293 or CHO cells) in the form of, for example, an Fc fusion (e.g., an FcLALA fusion).

[0165] In any of the above embodiments, preferably the shortened loop region has an amino acid length of 10, 9, 8, 7, 6, or less than 5, and preferably has an amino acid length of 7. More preferably, the mutation in the B'C' loop region includes: (a) Substitution of aa74~aa83 in the B'C' loop region, for example, substitution with a short B'C' loop sequence from a member of the IL family of 4-helix short-chain cytokines, such as the B'C' loop sequence of IL15, preferably substitution with the sequence GDASIH, or (b) Cleavage of aa74~aa83 in the B'C' loop region, for example, cleavage of 1, 2, 3 or 4 amino acids from the C-terminus, preferably cleavage to form the sequence (Q / G)SKN(F / I)H, more preferably cleavage to form a sequence selected from the following:

[0166] [Table 4]

[0167] Preferably, the mutation of the IL-2Rβγ bond interface includes the above mutation of the IL-2Rβγ bond interface. Preferably, the mutation of the IL-2Rα bond interface includes the above mutation of the IL-2Rα bond interface. Preferably, the mutant protein has improved characteristics, including (i) improved expression levels and / or protein purity (e.g., purity after one-step affinity chromatography by SEC-HPLC detection), and optionally (ii) weakened IL2Rβ binding and / or (iii) altered IL-2Ra binding.

[0168] In one embodiment, the method of the present invention further includes, after steps (1) and (2), a step of identifying the characteristics of the mutant protein, namely (i) the expression level and / or the purity of the purified protein (e.g., purity after one-step affinity chromatography by SEC-HPLC detection), and optionally (ii) weakened IL2Rβ binding and / or (iii) altered IL2Rα binding. In a further embodiment, the method of the present invention further includes, after steps (1) and (2), a step of identifying the characteristics of the mutant protein, namely (i) the expression level and / or the purity of the purified protein (e.g., purity after one-step affinity chromatography by SEC-HPLC detection), and (ii) weakened IL2Rβ binding and preferably maintained IL2Rα binding.

[0169] In one embodiment, the method includes a step of identifying IL-2 mutations with improved drug potential (e.g., expression level and / or product stability and / or homogeneity, e.g., one-step Fc affinity chromatography purity) before performing the mutation combination of step (1). In one preferred embodiment, the drug potential of the mutant protein is improved by substituting or cleaving the B'C'loop loop with a shortened B'C'loop loop.

[0170] In one embodiment, the method includes the step of identifying mutations at the IL-2Rβγ binding interface and / or mutations at the IL-2Rα binding interface that confer weakened IL-2Rβ binding and / or altered IL-2Ra binding ability compared to wild-type IL-2, before performing the combination of mutations in step (1).

[0171] As will be apparent to those skilled in the art, these mutations can be combined with mutations that confer further improved drug potential or other enhanced properties to obtain IL-2 mutant proteins with multiple enhanced properties.

[0172] In one embodiment, a mutation at the IL-2Rβγ binding interface resulting in weakening of (e.g., known) IL-2Rβγ binding is introduced into the IL-2 protein in combination with a mutation in the B'C' loop region that improves (e.g., known) drug potential, and then characterized. In one preferred embodiment, the characterization includes weakened IL-2Rβγ binding and improved drug potential (e.g., improved expression and / or purity, and / or product stability and / or homogeneity) compared to wild-type IL-2, and optionally, essentially unchanged or weakened or enhanced IL-2Rα binding affinity.

[0173] In some embodiments, the parental wild-type IL-2 protein used as the mutation template is preferably at least 85%, or at least 90% or 95%, identical to SEQ ID NO: 1, and more preferably a human-derived IL-2 protein.

[0174] 8. Pharmaceutical compositions and pharmaceutical preparations

[0175] The present invention further comprises compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an IL-2 mutant protein or its fusion, dimer, or immune complex, and compositions comprising polynucleotides encoding an IL-2 mutant protein or its fusion, dimer, or immune complex. These compositions may also optionally contain suitable pharmaceutically acceptable auxiliary materials, such as pharmaceutically acceptable carriers and pharmaceutically acceptable excipients, including buffers, that are known in the art.

[0176] 9. Combination Products

[0177] In one embodiment, the present invention further provides a combination product comprising the mutant protein of the present invention or its fusion, dimer, or immune complex, 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 therapeutic method of the present invention.

[0178] In some embodiments, the present invention provides a combination product in which the other therapeutic agent is, for example, an antibody, which is effective in stimulating an immune response to further enhance, stimulate, or upregulate the immune response of a subject.

[0179] In some embodiments, the combination product is used for the prevention or treatment of cancer. In some embodiments, the cancer is, for example, a cancer of the gastrointestinal tract, such as rectal cancer, colon cancer, or colorectal cancer. In some embodiments, the combination product is used for the prevention or treatment of infections, for example, bacterial infections, viral infections, fungal infections, or protozoan infections.

[0180] 10. Treatment methods and use

[0181] In this specification, the terms “individual” and “subject” may be used interchangeably and refer to mammals. Mammals include, but are not limited to, livestock (e.g., dairy cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats). In particular, the subject is human.

[0182] In this specification, the term “treatment” refers to a clinical intervention that seeks to alter the natural process of disease in the individual receiving treatment. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and improvement or mitigation of the prognosis.

[0183] In one embodiment, the present invention provides a method for stimulating the immune system of a subject, comprising the step of administering to the subject a pharmaceutical composition comprising an effective amount of the IL-2 mutant protein or fusion or dimer or immune complex of the present invention.

[0184] The weakened IL-2-Fc molecule of the present invention can effectively avoid the excessive release of inflammatory factors caused by strong stimulation of lymphocytes, and has more stable and longer-acting pharmacokinetic properties. Therefore, in one embodiment, a sufficiently high human drug exposure can be achieved with a relatively low single dose, avoiding drug-related toxicity due to excessively high Cmax.

[0185] Accordingly, in some embodiments, the present invention relates to a method for enhancing the immune response of a subject's body, comprising the step of administering to the subject an effective amount of any IL-2 mutant protein or its fusion or immune complex described herein. In some embodiments, the IL-2 mutant protein or its fusion or immune complex of the present invention is administered to a subject having a tumor to stimulate an anti-tumor immune response. In some other embodiments, an antibody or its antigen-binding moiety of the present invention is administered to a subject having an infection to stimulate an anti-infective immune response.

[0186] In another aspect, the present invention relates to a method of treating a disease in a subject, such as cancer, comprising administering to the subject an effective amount of any IL-2 mutant protein described herein, or a fusion or immunocomplex thereof. The cancer may be cancer at an early, middle or late stage or metastatic cancer. In some embodiments, the cancer may be a gastrointestinal cancer, such as rectal cancer, colon cancer, colorectal cancer, etc.

[0187] In another aspect, the present invention relates to a method of treating an infectious disease in a subject, such as chronic infection, comprising administering to the subject an effective amount of any IL-2 mutant protein described herein or a fragment thereof, or an immunocomplex, multispecific antibody, or pharmaceutical composition comprising the antibody or fragment. In one embodiment, the infection is a viral infection.

[0188] In another aspect, the present invention relates to a method of treating a Treg regulation-related disease in a subject, comprising administering to the subject an effective amount of any IL-2 mutant protein described herein or a fragment thereof, or an immunocomplex, multispecific antibody, or pharmaceutical composition comprising the antibody or fragment. In one embodiment, the disease is related to immune down-regulation mediated by IL-2-dependent Treg cells. In one embodiment, the disease is an autoimmune disease.

[0189] The mutant protein of the present invention (and pharmaceutical compositions containing the same or their fusions, dimers, or immune complexes, or any other therapeutic agent) may be administered by any suitable method, including parenteral administration, intrapulmonary administration, intranasal administration, and, if necessary for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. The method of administration is determined to some extent by whether the administration is short-term or long-term, and the administration may be by any suitable route, including injection, such as intravenous or subcutaneous injection. In this specification, however limited, various administration schedules include single doses or multiple doses at multiple times, bolus administration, pulse infusion, etc.

[0190] To prevent or treat a disease, the appropriate dose of the mutant protein of the present invention (when administered alone or in combination with one or more other therapeutic agents) is determined based on the type of disease being treated, the type of antibody, the severity and progression of the disease, whether the administration is for preventive or therapeutic purposes, past treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is administered appropriately to the patient in a single treatment or over a series of treatments.

[0191] In a further embodiment, the present invention also provides the use of the IL-2 mutant proteins, compositions, immune complexes, fusions, and dimer molecules of the present invention in the preparation of drugs used in the above-described methods (for example, for therapeutic purposes).

[0192] To aid in understanding the present invention, the following embodiments are described. It is not intended, nor should it be, to interpret the embodiments in any way as limiting the scope of the claims of the present invention. [Examples]

[0193] Example 1: Design and construction of IL-2Rα binding interface mutants of interleukin 2

[0194] Design and construction of an interleukin-2 mutation library

[0195] Design of a mutation library

[0196] Based on the crystal structure (PDB:1Z92) (shown in Figure 1) of the interleukin-2 (IL-2) and its α-receptor CD25 (IL-2Rα) complex, the IL-2 residues at the interaction site were mutated as shown in Table 1. Wild-type IL-2 (uniprot:P60568,aa21-153,C125S, IL-2 WT The (abbreviated as) was used as a template for constructing the mutation library. The original amino acids for each site accounted for 50%, and the remaining 50% was divided equally among the "mutant amino acids" in Table 1, using yeast-based IL-2 for the mutual binding sites of IL-2 and IL-2Rα, named IBYDL029 (Innoventbio Yeast Display Library). mutant A display library was designed and produced. Flow cytometry FACS was used to screen the library for IL-2 variants that do not bind to IL-2Rα.

[0197] [Table 5]

[0198] IL-2 WT The sequence is shown in Sequence ID No. 1 in this application, in which the C125S mutation is introduced at position 125 to avoid the formation of a disulfide-bridged IL-2 dimer.

[0199] According to existing literature, IL-2 mutant IL-2 3X It did not bind to IL-2Rα, and its binding affinity to IL-2Rβ remained constant (Rodrigo Vazquez-Lombardi et al, Nature Communications, 8:15373, DOI:10.1038 / ncomms15373). IL-2 3X It was displayed on the surface of yeast and used as a control. IL-2 3X The sequence is shown in SEQ ID NO: 4, and the protein in question is IL-2 WTSimilarly, this also includes the C125S mutation.

[0200] Identification of IL-2Rα binding interface mutants

[0201] IL-2 receptor expression and purification

[0202] Vector construction

[0203] The IL-2 receptors IL-2Rα (Uniprot:P01589,aa22-217) and IL-2Rβ (Uniprot:P14784,aa27-240) were constructed in the pTT5 vector (Addgene) by ligating an avi tag (GLNDIFEAQKIEWHE, this tag peptide can be biotinylated by the BirA enzyme) and six histidine tags (HHHHHH) to the C-terminus of each sequence. The sequences of the receptors produced by these constructions are shown in SEQ ID NOs. 5 and 6.

[0204] The IL-2 receptor βγ complex is an Fc heterodimer based on knobs in holes. The IL-2Rβ sequence is constructed at the N-terminus of the Fc-knob (SEQ ID NO: 7), and the IL-2Rγ sequence is constructed at the N-terminus of the Fc-hole (SEQ ID NO: 8). After being constructed in the pcDNA3.1 vector, they were co-expressed in Expi293 cells.

[0205] Plasmid transfection

[0206] Depending on the required transfection volume, Expi293 cells (Invitrogen) are passaged, and the cell density is increased to 1.5 × 10⁶ the day before transfection. 6 The concentration was adjusted to 10 cells / mL. On the day of transfection, the cell density was approximately 3 × 10⁶. 6It was cells / mL. Opti-MEM medium (Gibco product number: 31985-070) at 1 / 10 (v / v) of the final volume was used as the transfection buffer, and the expression plasmid constructed above was added, mixed uniformly, and filtered through a 0.22 μm filter head for use. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid in the previous step (the mass ratio of plasmid to PEI was 1:3), mixed uniformly, and then incubated at room temperature for 10 min to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently injected into HEK293 cells and mixed uniformly. After culturing at 37 °C under 8% CO2 for 24 h, valproic acid (VPA) (Sigma, product number: P4543-100G) was added until the final concentration reached 2 mM and 2% (v / v) of Feed (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone), and then cultured for an additional 6 days.

[0207] Purification of IL-2Rα-His and IL-2Rβ-His proteins

[0208] Before purification, the collected culture medium was centrifuged at 4500 rpm for 30 minutes to discard the cells. Next, the supernatant was filtered through a 0.22 μm filter. The nickel column used for purification (5 mL Histrap excel, GE, 17-3712-06) was immersed in 0.1 M NaOH for 2 hours, then rinsed with 5 to 10 times the column volume of ultrapure water to remove the alkaline solution. Before purification, the purification column was equilibrated with five times the column volume of binding buffer (20 mM Tris pH 7.4, 300 mM NaCl). The cell supernatant was passed through the equilibrated column, and then ten times the column volume of washing buffer (20 mM Tris pH 7.4, 300 mM NaCl, 10 mM imidazole) was passed through the column to remove nonspecifically binding heteroproteins. Subsequently, the target protein was eluted with three to five times the column volume of eluate (20 mM Tris pH 7.4, 300 mM NaCl, 100 mM imidazole). The collected proteins were concentrated by ultrafiltration and then replaced with PBS (Gibco, 70011-044). Further isolation and purification were performed using a Superdex 200 increase (GE, 10 / 300GL, 10245605), and monomer elution peaks were collected. PBS (Gibco, 70011-044) was used for column equilibration and elution buffering. A 100 μg sample of the purified protein was taken, and the protein purity was measured using a gel filtration chromatography column SW3000 (TOSOH product number: 18675).

[0209] Purification of IL-2 receptor βγ protein

[0210] After culturing the cells, they were centrifuged at 13,000 rpm for 20 minutes, the supernatant was collected, and the supernatant was purified using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). The procedure was as follows: Before purification, the packed column was equilibrated with five times the column volume of equilibration solution (0.2 M Tris, 1.5 M NaCl, pH 7.2). The collected supernatant was passed through the column, and the packed column was further washed with ten times the column volume of equilibration solution to remove nonspecifically bound proteins. The packing material was rinsed with five times the column volume of elution buffer (1 M sodium citrate, pH 3.5), the eluate was collected, the collected protein was concentrated by ultrafiltration and replaced with PBS (Gibco, 70011-044), and then further isolated and purified using superdex200 increase (GE, 10 / 300GL, 10245605). A single elution peak was collected, and the column equilibration and elution buffer were PBS (Gibco, 70011-044). 100 μg of the purified protein sample was taken, and the protein purity was measured using a gel filtration chromatography column SW3000 (TOSOH product number: 18675).

[0211] Biotinylation labeling of IL-2Rα, IL-2Rβ, and IL-2Rβγ proteins

[0212] The enzyme-catalyzed biotin labeling method is as follows: To each of the appropriate amounts of IL-2Rα, IL-2Rβ, and IL-2Rβγ protein solutions expressed and purified as described above, 1 / 10 (m / m) mass of His-BirA protein (uniprot: P06709) was added. Simultaneously, 2 mM ATP (sigma product number: A2383-10G), 5 mM MgCl2, and 0.5 mM D-biotin (AVIDITY product number: K0717) were added, and the mixture was incubated at 30°C for 1 hour. The mixture was then purified using a Superdex200 increase (GE, 10 / 300GL, 10245605) to remove excess biotin and His-BirA. The purified sample was then validated using a Fortebio Streptavidin (SA) sensor (PALL, 18-5019) to confirm the success of the biotin labeling.

[0213] IL-2 mutant - Expression, purification, and identification of FC fusion proteins

[0214] Construction of expression plasmids

[0215] Using flow cytometry (FACS), yeast-based IL-2 mutant IL-2Rα not coupled from display library IBYDL029 mutant Screening was performed on IL-2. mutant - IL-2 was screened from the library to express FC fusion proteins. mutant The sequence was ligated to FcLALA via two GGGGS sequences and then constructed into a pCDNA3.1(Addgene) vector.

[0216] For comparison, IL-2 WT -FC and IL-2 3X - To express FC fusion protein, IL-2 WT IL-2 3X The gene sequence was linked to FcLALA via two GGGGS sequences and constructed on pCDNA3.1. In this example, Fc refers to the Fc (abbreviated as FcLALA, sequence number 12) of human IgG1 having mutations L234A and L235A.

[0217] Expression and purification of IL-2 and FC fusion proteins

[0218] A vector containing the gene encoding the fusion protein was introduced into HEK293 cells by chemical transfection. HEK293 cells cultured according to the manufacturer's protocol were transiently transfected using the chemical transfection reagent PEI. First, plasmid DNA and transfection reagent were prepared in an ultraclean bench. 3 mL of Opti-MEM medium (Gibco product number: 31985-070) was placed in a 50 mL centrifuge tube, 30 μg of the corresponding plasmid DNA was added, and the Opti-MEM medium containing the plasmid was filtered through a 0.22 μm filter head. Subsequently, 90 μg of PEI (1 g / L) was added, and the mixture was allowed to stand for 20 minutes. The DNA / PEI mixture was slowly injected into 27 mL of HEK293 cells and mixed uniformly. After culturing for 20 hours at 37°C under 8% CO2 conditions, VPA was added until the final concentration reached 2 mM and 2% (v / v) feed, and the cells were cultured for another 6 days.

[0219] After cell culture, the cells were centrifuged at 13000 rpm for 20 minutes, the supernatant was collected, and the supernatant was purified using a pre-packed Hitrap Mabselect Sure column. The procedure was as follows: Before purification, the packed column was equilibrated with five times the column volume of equilibrium solution (0.2 M Tris, 1.5 M NaCl, pH 7.2), the collected supernatant was passed through the column, and the packed column was further washed with ten times the column volume of equilibrium solution to remove nonspecifically bound proteins. The packing material was rinsed with five times the column volume of elution buffer (1 M sodium citrate, pH 3.5), the eluate was collected, 80 μL of Tris (2 M Tris) was added per 1 mL of eluate, and the solution was replaced with PBS buffer using an ultrafiltration concentrator, and the concentration was measured. 100 μg of the purified protein was adjusted to a concentration of 1 mg / mL, and the purity of the IL-2-Fc dimer protein was measured using a gel filtration chromatography column. The results are shown in Table 2. Figure 3 shows the sequences of the IL-2 variants of the present invention listed in Table 2.

[0220] [Table 6]

[0221] IL-2 mutant - Measurement of affinity between FC and its receptor

[0222] Using the Biolayer Interferometry (ForteBio) method, the present invention's IL-2 mutant -FC and its receptor equilibrium dissociation constant (K D ) was measured.

[0223] ForteBio affinity measurement was performed according to the conventional method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p.270-8). In short, candidate IL-2 mutant -The affinity of FC to IL-2Rα and IL-2Rβ was measured as follows: The sensor was equilibrated offline with analytical buffer for 20 minutes, then detected online for 120 seconds to establish a baseline. Human biotinylated IL-2Rα or IL-2Rβ was loaded onto the SA sensor (PALL, 18-5019) and ForteBio affinity measurements were performed. The sensor loaded with biotinylated IL-2Rα or IL-2Rβ was subjected to 100 nM IL-2 until a plateau was reached. mutant After being placed in a solution containing -FC, the sensor was transferred to the analytical buffer and dissociated for at least 2 minutes to measure the binding and dissociation rates. Dynamical analysis was performed using a 1:1 binding model.

[0224] In the experiment performed using the above measurement method, IL-2 expressed in HEK293-F mutant -FC and its affinity K for its receptor D The values ​​are shown in Table 3. For control, IL-2 measured by the same method is also shown. WT -FC and IL-2 3X - Affinity K of FC fusion proteins D The values ​​are also shown in Table 3.

[0225] [Table 7]

[0226] As can be seen from the affinity data, all of the above mutants obtained from the IBYDL029 library block binding to IL-2Rα.

[0227] Example 2: Construction, screening, and identification of IL-2 chimeras and cleavage mutants

[0228] Design of IL-2 B'C'loop Chimera and Severed Body

[0229] B'C'loop: This is the linking sequence of the B helix and C helix of IL-2 (Figure 2A), and contains a total of 11 amino acids from A73 to R83.

[0230] A comparison of the crystal structures of the IL-2 monomer (PDB:1M47) and its composite (PDB:2ERJ) revealed that the B'C' loop is highly active in solution and cannot form a relatively stable conformation, resulting in the absence of the B'C' loop in the crystal structure of the IL-2 monomer.

[0231] By genetically modifying the B'C'loop, we improved its stability, which in turn improved the stability of IL-2. Therefore, we aligned it with the human IL15 crystal structure (PDB:2Z3Q) and found that its B'C'loop was short and stable (Figure 2B). Accordingly, we designed one IL-2 chimeric molecule (L017) and four cleavage molecules (L057-L060) (shown in Table 4).

[0232] [Table 8]

[0233] Construction of expression plasmids

[0234] Wild type IL-2(uniprot:P60568,aa21-153,C125S,IL-2WT (abbreviated as IL-2), and IL-2 variant IL-2 3X (R38D, K43E, E61R), B'C'loop chimeric and cleaved proteins were linked to human IgG1 Fc (L234A, L235A, abbreviated as FcLALA, SEQ ID NO: 12) via GSGS linkage sequences, and constructed in a pTT5 vector, expressing the following proteins:

[0235] [Table 9]

[0236] B'C'loop chimeric forms (Y017) or cleaved forms (Y057) were screened from a library to obtain mutant Y30E1 (K35E, T37E, R38E, F42A), which were then linked to FcLALA via two GGGGS links and constructed into a pCDNA3.1 vector to express the following proteins. Here, Y092 has the chimeric B'C'loop loop sequence AGDASIH, and Y144 is based on Y092 with increased T3A amino acid substitutions to remove the N-terminal O-glycosyl modification of IL-2.

[0237] [Table 10]

[0238] Note that IL-2 WT and IL-2 3X The following proteins were expressed by linking them to FcLALA via two GGGGS molecules and constructing them in a pCDNA3.1 vector.

[0239] [Table 11]

[0240] The specific sequence information for the above protein molecules is shown in the sequence listing.

[0241] Expression and purification of fusion proteins

[0242] The above protein molecules were expressed in HEK293 cells and CHO cells, respectively. Expression in HEK293 cells was performed according to the method of Example 1 used for the expression of the IL-2-Fc fusion protein. Expression in CHO cells was performed as follows.

[0243] Depending on the required cell volume, ExpiCHO cells (Invitrogen) are passaged, and the cell density is increased to 3.5 × 10⁶ the day before transfection. 6 The cell density was adjusted to 10 cells / mL. Cell density (approximately 8-10 × 10) was measured on the day of transfection. 6 Individual cells (per mL) were detected, and the survival rate reached over 95%. ExpiCHO TM Cell density 6 × 10⁶ using Expression Medium (Gibco product number: A29100-01) 6 Prepared to the number of cells / mL. 8% (v / v) of the final volume of OptiPRO. TM Using SFM (Gibco product number: 12309-019) as the transfection buffer, add the corresponding amount of plasmid (0.8 μg / mL cells), mix uniformly, filter through a 0.22 μm filter membrane to remove bacteria, and then inoculate with ExpiFectamine at a cell ratio of 3.2 μL / mL. TM The reagent from the CHO Transfection Kit (Gibco, product number: A29130) was added. The complex formed by the transfection reagent and plasmid DNA was incubated at room temperature for 1-5 minutes, then gradually added to the cells. After 18 hours of incubation at 37°C under 8% CO2 conditions, 0.6% (v / v) enhancer and 30% (v / v) feed were added, and the cells were incubated for another 6 days.

[0244] After culturing the cells, the cell culture medium was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected. The supernatant was purified using a pre-packed column Hitrap Mabselect Sure (GE, 11-0034-95). The procedure was as follows: Before purification, the packed column was equilibrated with five times the column volume of equilibrium solution (20 mM Tris, 150 mM NaCl, pH 7.2). The collected supernatant was passed through the column, and the packed column was further washed with ten times the column volume of equilibrium solution to remove nonspecifically binding proteins. The packing material was rinsed with five times the column volume of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. 80 μL of Tris (2 M Tris) was added per 1 mL of eluate, and the solution was replaced with PBS buffer (Gibco, product number: 70011-044) using an ultrafiltration concentrator (MILLIPORE, product number: UFC901096), and the concentration was measured. 100 μg of purified protein was adjusted to a concentration of 1 mg / mL, and the purity of the protein was measured using a gel filtration chromatography column SW3000 (TOSOH product number: 18675).

[0245] The fusion proteins of the B'C'loop chimeric form (Y017) and cleaved forms (Y057 / 058 / 059) showed significantly higher expression levels and one-step affinity chromatography purity in HEK293 cells compared to the wild-type IL-2 fusion protein (Y001). The results are shown in Table 5 below.

[0246] [Table 12]

[0247] Fusion proteins containing B'C'loop chimeric forms (Y092 and Y144), cleaved forms (Y089), and further mutant Y30E1 showed significantly improved expression levels in CHO cells and one-step affinity chromatography purity compared to the wild-type IL-2 fusion protein (Y045). The results are shown in Table 6 below.

[0248] [Table 13]

[0249] As can be seen from the data above, 1) point mutant molecules such as Y30E1 screened from the yeast library can block binding to IL-2Rα. 2) B'C'loop chimeric molecules and cleavage molecules increased the expression level and purity of the molecule. 3) Combination molecules of Y30E1 and B'C'loop mutants blocked IL-2Rα and improved the expression level and purity of the molecule.

[0250] In vitro functional experiments of mutants

[0251] Each IL-2 mutant - The first Human CD8 by FC + By detecting the activation of p-STAT5 signaling in T cells, the CD25 of each mutant can be identified. + Cells and CD25 - Activation of cells was confirmed. The specific steps were as follows:

[0252] 1. Resuscitation of PBMC cells: a) PBMC cells (Allcells product number: PB005F, 100M pack) were removed from liquid nitrogen and quickly placed in a 37°C water bath to revive the PBMC cells. b) Cells were added to 10 mL of X-VIVO15 (Lonza product number: 04-418Q) medium containing preheated 5% human AB serum (GemCell product number: 100-512) and 1‰ of DNA enzyme (STRMCELL product number: 07900), centrifuged at 400G and 25°C for 10 minutes (all subsequent centrifugations were performed under these conditions), and washed once. c) The cells were resuspended in 20 mL of culture medium and incubated overnight in a 37°C carbon dioxide incubator.

[0253] 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), 100 μL of human AB serum, and 100 μL of human CD8 + T cell purification kit (Invitrogen product number: 11348D), and the antibody mixture was negatively screened and the cells were resuspended. c) After mixing uniformly, it was incubated at 4 °C for 20 min and shaken once every 5 min. d) After incubation, 10 mL of Robosep buffer was added and centrifuged for two washes. e) At the same time, 7 mL of Robosep buffer was added to 1 mL of magnetic microspheres (human CD8 + T cell purification kit), placed on a magnetic stand for 1 min, and the supernatant was discarded to pre-wash the magnetic microspheres. f) 1 mL of Robosep buffer was added to each to resuspend the microspheres and cells respectively. After mixing uniformly, it was incubated with rotation at room temperature for 30 min. g) After incubation, 6 mL of Robosep buffer was added, placed on a magnetic stand for 1 min, and the supernatant was collected. h) The collected liquid was placed on a magnetic stand for 1 min again, and the supernatant was collected. i) Centrifuged to discard the supernatant, resuspended with pre-warmed T medium, and adjusted the density to 1×10 6 / mL. j) One-third of the cells were collected and, if necessary later, CD25 expression was stimulated. The remaining cells were placed in a 37 °C carbon dioxide incubator and left standing for overnight culture.

[0254] 3. CD8 + T cells were stimulated to express CD25: a) To one-third of the CD8 + T cells purified in step 2, magnetic microspheres of anti-human CD3 / CD28 antibody (GIBCO product number: 11131D) were added, and the ratio of cells to microspheres was 3:1. b) Placed in a 37 °C carbon dioxide incubator and left standing for 3 days. c) 10 mL of medium was added and washed twice. d) Add the medium, adjust until the cell density reaches 1×10 6 / mL, place it in a carbon dioxide incubator at 37°C, let it stand still, and culture for 2 days.

[0255] 4. Detection of cell purity and expression level: a) Detect CD8 and CD25 of the cells 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) The cells in step 2 are CD8 + CD25 - T cells, and the cells in step 3 are CD8 + CD25 + T cells.

[0256] 5. Detection of EC mutant of p-STAT5 signal activation in CD8 + CD25 - T cells by each IL-2 50 -FC: a) Seed CD8 + CD25 - T cells at 1×10 5 cells per well in a 96-well U-bottom culture plate (Costar product number: CLS3799-50EA). b) Add 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, start from the highest concentration of 266.7 nM and dilute in a 4-fold gradient, with a total of 12 gradients, and incubate in an incubator at 37°C for 20 min. c) Add 55.5 μL of 4.2% formaldehyde solution and fix at room temperature for 10 min. d) Centrifuge to discard the supernatant, add 200 μL of ice-cold methanol (Fisher product number: A452-4) to resuspend the cells, and incubate in a refrigerator at 4°C for 30 min. e) The supernatant was centrifuged and washed three times with 200 μL of staining buffer (BD product number: 554657). f) Add 200 μL of permeabilization / fixation buffer (BD product number: 51-2091KZ) containing anti-p-STAT5-AlexFlour647 (BD product number: 562076, 1:200 dilution) and incubate at room temperature in the dark for 3 hours. g) The cells were washed three times with staining buffer, resuspended in 100 μL of staining buffer, and detected by flow cytometry. h) The EC of the p-STAT5 signal is plotted with the concentration of IL-2 molecules on the x-axis and the AlexFlour647 intermediate fluorescence value on the y-axis. 50 Values ​​are generated, and the results are shown in Table 7 below.

[0257] 6. Each IL-2 mutant - FC CD8 + CD25 + EC of p-STAT5 signaling activation in T cells 50 Detection: a) CD8 + CD25 + T cells are 1 × 10⁶ per well 5 The cells were placed in a 96-well U-bottom culture plate. b) Similar to bh in step 5, the EC of the p-STAT5 signal 50 Values ​​are generated, and the results are shown in Table 7.

[0258] [Table 14]

[0259] Example 3: Design of an IL-2 weakened mutant

[0260] Design of IL-2 weakening molecules

[0261] We further improved the pharmacokinetics (PK) of the IL-2 variant, extending its half-life while simultaneously reducing its toxicity. Based on the analysis of the crystal structure of the IL-2 complex (Figure 4) (PDB:2ERJ), we selectively mutated amino acids at the contact interface between IL-2 and IL-2Rβ, thereby reducing their affinity for each other.

[0262] Based on amino acid mutations at the binding interface of IL-2Rα, optimization of the IL-2B'C' loop, and amino acid mutations at the binding interface of IL-2Rβγ, and validated based on two format formats, the molecular designs are shown in Table 8. In some cases, these molecules may further include the T3A mutation to remove the O-glycosyl modification at the N-terminus of IL2.

[0263] [Table 15]

[0264] Based on the above design, in order to express and produce a Format 1 molecule, IL-2 having the following specific sequence structure (from N-terminus to C-terminus) is required. mutant Built: - IL-2 mutant sequence having amino acid sequence numbers 37-638, -Linker array GGGGSGGGGS, - The Fc sequence of sequence number 12.

[0265] Based on the above design, in order to express and produce Format 2 molecules, IL-2 having the following specific sequence structure (from N-terminus to C-terminus) is required. mutant - Built Fc.Knob and Fc.Hole: (1) IL-2 mutant -Fc.Knob - IL-2 mutant sequence having amino acid sequence numbers 37-638, -Linker array GGGGSGGGGS, -Fc.Knob sequence of sequence number 9, and (2) Fc. Hole -The Fc.Hole sequence of sequence number 10.

[0266] Expression and purification of IL-2 weakened mutants and measurement of their affinity to the receptor

[0267] Format 1 involved ligating the IL-2 mutant to the N-terminus of IgG1FcLALA via two G4S links to construct a bivalent IL-2 mutant molecule. Format 2 was a heterodimer based on Knob-in-hole IgG1FcLALA, where the IL-2 mutant molecule was ligated to the N-terminus of the IgG1FcLALA-Knob via two G4S links to construct a monovalent IL-2 mutant molecule. All sequences were constructed in the pcDNA3.1 vector.

[0268] Protein expression and purification: The experimental method was the same as in Example 2.

[0269] The equilibrium dissociation constant (KD) of the above-mentioned IL-2 variant and its receptor were measured using the biolayer interference (ForteBio) assay method.

[0270] ForteBio affinity measurements were performed according to a conventional method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013. 5(2): p.270-8). Affinity measurements of candidate molecules to IL-2Rα, IL-2Rβ, and IL-2Rβγ were performed: the sensor was equilibrated offline in analytical buffer for 20 minutes, then detected online for 120 seconds to establish a baseline. Human biotinylated IL-2Rα, IL-2Rβ, or IL-2Rγ were loaded onto an SA sensor (PALL, 18-5019) and ForteBio affinity measurements were performed. After placing the IL-2 receptor-loaded sensor in a solution containing 100 nM of IL-2 mutant molecules until a plateau was reached, the sensor was transferred to analytical buffer and dissociated for at least 2 minutes to measure binding and dissociation rates. Dynamical analysis was performed using a 1:1 binding model.

[0271] The experimental results are shown in Table 9. Compared to the wild type (Y045), the IL-2 mutant showed improved expression levels and purity, and simultaneously, the introduction of amino acid mutations at the IL-2Rβγ binding interface reduced the affinity for IL-2Rβ and IL-2Rβγ to varying degrees. The molecular structures in the table below are shown in Figure 6.

[0272] [Table 16-1] [Table 16-2] Note: "-" indicates not detected, and "NB" indicates unbound.

[0273] Example 4: In vitro functional experiment of IL-2 mutant

[0274] Detection of pSTAT5 signaling in IL-2 variants in human T lymphocytes

[0275] The binding of IL-2 to the IL-2 receptor on the surface of T cells activates the JAK-STAT signaling pathway in human T lymphocytes, where the STAT5 phosphorylation level was an important indicator for evaluating the level of activation of this signaling pathway. Normal T lymphocytes did not fundamentally express IL-2Rα. By detecting the pSTAT5 signaling of IL-2 mutant molecules against T lymphocytes, we evaluated the strength of the human T cell activation activity of different IL-2 mutant molecules.

[0276] Experimental materials and methods:

[0277] Experimental method:

[0278] 1. Resuscitation of PBMC cells

[0279] (1) PBMC cells (All Cells, product number PB004F-C, number LP191011A / LP190529) were cryopreserved in liquid nitrogen and thawed quickly by shaking at 37°C.

[0280] (2) The cells were slowly added to 10 mL of CTS medium (Gibco, product number A3021002, lot number 1989823, preheated to 37°C and containing 100 μl of DNA enzyme).

[0281] (3) The mixture was centrifuged at 300g / 8min and the supernatant was removed.

[0282] (4) The mixture was resuspended in 10 mL of CTS and transferred to a T75 culture flask (NUNC, product no. 156499), and stabilized overnight in a 5% incubator at 37°C.

[0283] 2. pSTAT5 experiment

[0284] (1) Culture the suspended cells overnight in PBMC, then spread the cells on a 96-well U-shaped plate (Cornning, product number CLS3799-50EA), with a cell count of 5 × 10 5 The value was cells / well.

[0285] (2) Different diluted detection antibodies were added to 96-well plates, and the detection antibodies were incubated with cells at 37°C for 30 minutes.

[0286] (3) The mixture was centrifuged at 400g / 5min and the supernatant was removed.

[0287] (4) Add 4% tissue cell fixative (Solarbio, product number: P1110) to 200 ul / well and centrifuge at room temperature at 400 g / 30 min.

[0288] (5) Add permeabilization solution (BD, product number: 558050) at a rate of 200 ul / well, allow to stand at 4°C for 30 minutes, and then centrifuge at 400 g / 10 min.

[0289] (6) The cells were washed a total of two times with 200 ul / well of perm / wash buffer (BD, product number 558050, lot number 7271605).

[0290] (7) Antibody staining solutions were prepared. The amount of pSTAT5 antibody (BD, product number 562076, lot number 9141872) was 3 ul / 100 ul of perm / wash buffer / well, and the amounts of BV421 anti-human CD3 antibody (Biolegend, product number 300434, lot number: B271302), FTIC anti-human CD4 antibody (Biolegend, product number 300506, lot number: B283935), and AF700 anti-human CD8a (Biolegend, product number 300924, lot number: B253967) were each 1 ul / 100 ul of perm / wash buffer / well. The solutions were incubated at room temperature for 1.5 hours and washed twice with perm / wash buffer.

[0291] (8) The sample was resuspended in 150 µl of perm / wash buffer per well and flow cytometry was performed.

[0292] Experimental results:

[0293] The results of detecting the pSTAT5 efficacy of IL-2 mutant molecules in normal T lymphocytes (CD4+ T cells, CD8+ T cells) are shown in Figures 7A, 7B, 7C, 7D, and 7E.

[0294] Phosphorylation levels in CD4+ and CD8+ T cells represented cell proliferative capacity and cell activity. As can be seen from Figure 7 and Table 10, one or more amino acids in the mutant IL-2Rβ binding interface reduced the phosphorylation levels of T cells in IL-2 mutants to varying degrees compared to the unmutated molecules Y-144 (format 1) and 2688 (format 2) of the IL-2Rβ binding interface.

[0295] [Table 17]

[0296] Detection of pSTAT5 signaling in IL-2 variants in activated T lymphocytes

[0297] IL-2 binding to the IL-2 receptor on the surface of T cells activates the JAK-STAT signaling pathway in T lymphocytes, where STAT5 phosphorylation levels were an important indicator for assessing the degree of activation of this signaling pathway. After T lymphocyte activation, the abundance of IL-2Rα(CD25) on the surface of T cells significantly increased.

[0298] Experimental method:

[0299] 1. Resuscitation of PBMC cells

[0300] (1) PBMC cells (All Cells, product number PB004F-C, number LP191011A) were cryopreserved in liquid nitrogen and thawed quickly by shaking at 37°C.

[0301] (2) The cells were slowly added to 10 mL of CTS medium (Gibco, product number A3021002, lot number 1989823, preheated to 37°C and containing 100 μl of DNA enzyme).

[0302] (3) The mixture was centrifuged at 300g / 8min and the supernatant was removed.

[0303] (4) The mixture was resuspended in 10 mL of CTS and transferred to a T75 culture flask (NUNC, product no. 156499), and stabilized overnight in a 5% incubator (Thermo, product no. 3111) at 37°C.

[0304] 2. Activation and quiescence of T lymphocytes

[0305] (1) Suspension cells were taken from PBMCs cultured overnight, the cells were counted, and an amount of CD3 / CD28 beads (Invitrogen, product number 11131D, lot number: 00783216) equivalent to the number of cells was added and activated for 48 hours to stimulate the cells.

[0306] (2) The beads and culture medium were removed, and the activated cells were washed.

[0307] (3) The activated cells were transferred to a T75 culture flask and kept still at 37°C and 5% for 48 hours.

[0308] 3. pSTAT5 experiment

[0309] (1) Place the cells in a 96-well U-shaped plate (Cornning, product number CLS3799-50EA), with a cell count of 5 × 10 5 The value was cells / well.

[0310] (2) Different diluted detection antibodies were added to 96-well plates, and the detection antibodies were incubated with cells at 37°C for 30 minutes.

[0311] (3) The mixture was centrifuged at 400g / 5min and the supernatant was removed.

[0312] (4) Add 4% tissue cell fixative (Solarbio, product number: P1110) to 200 ul / well and centrifuge at room temperature at 400 g / 30 min.

[0313] (5) Add permeabilization solution (BD, product number: 558050) to 200 ul / well, let stand at 4°C for 30 min, and centrifuge at 400 g / 10 min.

[0314] (6) The cells were washed a total of two times with 200 ul / well of perm / wash buffer (BD, product number 558050, lot number 7271605).

[0315] (7) Antibody staining solutions were prepared. The amount of pSTAT5 antibody (BD, product number 562076, lot number 9141872) was 3 ul / 100 ul of perm / wash buffer / well, and the amounts of BV421 anti-human CD3 antibody (Biolegend, product number 300434, lot number: B271302), FTIC anti-human CD4 antibody (Biolegend, product number 300506, lot number: B283935), and AF700 anti-human CD8a (Biolegend, product number 300924, lot number: B253967) were each 1 ul / 100 ul of perm / wash buffer / well. The solutions were incubated at room temperature for 1.5 hours and washed twice with perm / wash buffer.

[0316] (8) The sample was resuspended in 150 µl of perm / wash buffer per well and flow cytometry was performed.

[0317] Experimental results

[0318] CD8 + T cells, CD4 + CD25 - T cells, NK cells (CD3 - CD56 + ) and Treg cells (CD3 + CD4 + CD25 + The results of detecting the pSTAT5 efficacy of IL-2 mutant molecules in ) are shown in Figures 8A-G and Tables 11-12. As can be seen from the results, the pSTAT5 activity of the IL-2 mutants in this study against lymphocytes was weaker than that of human wild-type IL-2 (rhIL-2, R&D systems, product number: 202-IL), and at the same time, compared to other lymphocytes, the IL-2 mutant molecules in this study showed very high selectivity for Treg cells.

[0319] [Table 18]

[0320] [Table 19]

[0321] Example 5: In vivo antitumor effect of IL-2 mutant molecule

[0322] To demonstrate the in vivo antitumor effect of IL-2 mutant molecules, MC38 cells were inoculated into C57 mice, and the antitumor effect of the IL-2 mutant molecules of the present invention (Y144, 2113, 2162) was measured (the structures of molecules 2113 and 2162 are shown in Figure 6). SPF-grade female C57 mice (14-17g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used for the experiment, and the certificate of conformity number was NO.110011201109499961.

[0323] MC38 cells were regularly subcultured and used in subsequent in vivo experiments. Cells were collected by centrifugation, dispersed in PBS (1×), and the cell concentration was 5×10⁶. 6 A cell suspension was prepared at a concentration of 100 cells / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank of C57 mice to establish an MC38 tumor-bearing mouse model.

[0324] Seven days after tumor cell inoculation, the tumor volume of each mouse was detected and the mice were divided into groups (6 mice per group). The dosage and administration method are shown in Table 14. The treatment was administered on days 7, 14, and 21 after inoculation, and the tumor volume and body weight of the mice were monitored 2-3 times per week, as shown in Figures 9A and 9B. Monitoring was discontinued after 25 days. On day 25 after inoculation, the relative tumor inhibition rate (TGI%) was calculated. The formula was TGI% = 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-treatment tumor volume). Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L × W 2 The calculation was performed according to / 2. Body weight was measured using an electronic balance.

[0325] [Table 20]

[0326] The detection of tumor volume is shown in Figure 9A. On day 25 after inoculation, the monotherapy inhibition rates of the weakened IL-2 molecules 2113 (3 mg / kg) and 2162 (3 mg / kg) were 71.4% and 33.8%, respectively, compared to h-IgG. The results indicated that the modified IL-2 molecules (2113, 2162) had antitumor activity and exhibited a dose-response effect. Simultaneously, mouse body weight was detected, and the results are shown in Figures 9B and 9C. There was no significant difference in body weight between the mice in the 2113 (1 mg / kg), 2162 (1 mg / kg), and 2162 (3 mg / kg) groups. In contrast, the mice administered with the unweakened molecule Y144 showed intolerance and died before the end of the 25-day monitoring period.

[0327] In another MC38 tumor model, the in vivo antitumor effects of several other IL-2 mutant molecules (Y045, 2478, 2454) were demonstrated (the structures of molecules 2478 and 2454 are shown in Figure 6). For this experiment with MC38 tumor-bearing mice, SPF-grade female C57 mice (14-17g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) with certificate number 110011201109499826 were used. The experiment was basically performed as described above.

[0328] Seven days after tumor cell inoculation, the tumor volume of each mouse was detected and the mice were divided into groups (7 mice per group). The dosage and administration method are shown in Tables 13 and 14. The treatment was administered on days 7, 14, and 21 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 10A and 10B. Monitoring was discontinued after 25 days. On day 25 after inoculation, the relative tumor-to-graft (TGI%) was calculated. The formula was TGI% = 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-treatment tumor volume). Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L × W 2 The calculation was performed according to / 2. Body weight was measured using an electronic balance.

[0329] [Table 21]

[0330] The tumor suppression rates are shown in Table 15. On day 25 after inoculation, the monotherapy suppression rates for molecules Y045 (1 mg / kg), 2478 (1 mg / kg), 2454 (1 mg / kg), Y045 (3 mg / kg), 2478 (3 mg / kg), and 2454 (3 mg / kg) were 5.3%, 39.1%, 15.6%, 19.8%, 52.6%, and 23.0% respectively, compared to h-IgG. The results showed that the unmodified IL2 molecule (Y045) had both antitumor activity and a dose-dependent effect, while the modified weakened IL2 molecules (2478, 2454) also had both antitumor activity and a dose-dependent effect, but their efficacy was superior to that of the unmodified IL2 molecule (Y045). Simultaneously, mouse body weight was detected, and the results are shown in Figures 10B and 10C. There was no significant difference in mouse body weight.

[0331] [Table 22]

[0332] Example 6: In vivo study of IL-2 variant molecules in autoimmunity

[0333] To demonstrate the potential of the IL-2 attenuating molecule in autoimmunity, blood samples were collected from C57 mice before administration and on days 3 and 7 after administration to measure the effect of the IL-2 attenuating molecule of the present invention on immune cells. SPF-grade female C57 mice (14-17g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used for the experiment, and the certificate of conformity number was NO.110011201109500076.

[0334] Thirty C57 mice were randomly divided into groups (6 mice per group), and the dosage and administration method are shown in Table 16. A single dose was administered, and the mice's body weight was monitored twice a week as shown in Figures 11A and 11B, with monitoring ending after 7 days. Blood samples were taken from the mice before administration and on days 3 and 7 after administration to measure CD45 levels. + Total T lymphocyte count, including Treg, NK, CD4T conv (CD4+Foxp3-), and CD8 in the blood. + The changes in the proportion of T cells were detected and are shown in Figures 11c, 11d, 11e, and 11f.

[0335] The molecule being measured, 2602, is wild-type IL-2. WT The weakening molecules 3079, 3055, and 3082, which contain sequence number 1, are all Format 2 IL-2-Fc dimer molecules and are shown in Figure 6.

[0336] As the results show, the IL-2 mutant molecule in this study weakens its binding to IL-2Rβ, thereby stimulating the proliferation of pro-inflammatory NK cells by the IL-2 mutant molecule, wild-type IL-2 molecule 2602 (IL-2 WT Although weaker than the previous mutation, the amplification of Treg cells, which are still immunosuppressive cells, was maintained, and the extent of proliferation of the IL-2 mutant molecule against Treg cells was similar. Furthermore, the IL-2 mutant molecule did not produce any significant proliferative effect on CD4T convolutional cells or CD8T cells. These results suggest that mutating the binding site of IL-2 and IL-2Rβ can significantly improve selectivity for Treg cells and other immune cells.

[0337] [Table 23]

[0338] Explanation of the sequence list

[0339] [Table 24-1] [Table 24-2]

[0340] A detailed description of the IL-2 dimer molecule constructed by the present invention is shown in the table below. Here, the "IL-2 sequence SEQ ID NO" column shows the amino acid sequence of the IL-2 portion of the molecule, and the "Notes on the IL-2 portion" column explains that the IL-2 amino acid sequence of the molecule consists of the SEQ ID NO: amino acid sequence having the mutation, based on the amino acid sequence in the "SEQ ID NO:" column and the mutation in the "Mutation" column. "Format 1" indicates that the molecule is a homodimer, formed by sequentially linking each monomer (from the N-terminus to the C-terminus) with the indicated IL-2 amino acid sequence, linker sequence (G4S) 2, and SEQ ID NO. 12. "Format 2" indicates that the molecule is a heterodimer, formed by sequentially linking one monomer (from the N-terminus to the C-terminus) with the indicated IL-2 amino acid sequence, linker sequence (G4S) 2, and SEQ ID NO. 9, and the other monomer is SEQ ID NO. 10.

[0341] [Table 25-1] [Table 25-2]

[0342] [Table 26-1] [Table 26-2] [Table 26-3]

[0343] [Table 27-1] [Table 27-2]

[0344] Table 28

Claims

1. IL-2 mutant protein, The aforementioned mutant protein exhibits the following mutations compared to wild-type human IL-2: (a) A shortened B'C' loop region and mutations at the IL-2Rβγ binding interface of IL-2; (b) a shortened B'C' loop region, a mutation at the IL-2Rβγ binding interface of IL-2, and a mutation at the IL-2Rα binding interface of IL-2; or (c) Mutations of IL-2 at the IL-2Rβγ binding interface and mutations of IL-2 at the IL-2Rα binding interface Includes, The shortened B'C' loop region has a sequence located between amino acid residues aa72 and aa84, and the sequence is shortened to a length of less than 10 amino acids, and The shortened B'C' loop region is, (i) The sequence of GDASIH at positions aa74 to aa83; or, (ii) The sequence (Q / G)S(K / A / D)N(F / I)H at positions aa74 to aa83; Includes, The mutations at the IL-2Rβγ binding interface are as follows: The mutations include those selected from the group consisting of D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I; The mutations at the IL-2Rα binding interface include the combination of K35E + T37E + R38E + F42A, or the combination of K35E + T37E + R38E; Here, the positions of the amino acids are numbered according to Sequence ID No. 1; The wild-type human IL-2 comprises the sequence of sequence number 1, 2, or 3, The IL-2 mutant protein has at least 87%, 88%, 89%, or 90% identity with wild-type human IL-2. The aforementioned IL-2 mutant protein.

2. The mutation at the IL-2Rβγ binding interface is N88R, as described in claim 1, for the IL-2 mutant protein.

3. The mutant protein according to claim 1 or 2, wherein the mutation at the IL-2Rα binding interface includes a combination of K35E + T37E + R38E + F42A.

4. The mutant protein according to any one of claims 1 to 3, wherein the IL-2 mutant protein includes a loop region having a sequence from aa74 to aa83 selected from the group consisting of GDASIH, QSKNFH, GSKNFH, QSANFH, and QSANIH.

5. The aforementioned IL-2 mutant protein, (i) B'C' loop region sequences located between amino acid residues aa72 and aa84: AGDASIH, AQSKNFH, or SGDASIH; and, (ii) A mutation at the IL-2Rβγ binding interface selected from the group consisting of N88R, D20N, and N88D, The mutant protein according to claim 1, comprising:

6. The aforementioned IL-2 mutant protein, (i) the B'C' loop region sequence of AGDASIH located between amino acid residues aa72 and aa84; and (ii) Mutation N88R at the IL-2Rβγ binding interface, The mutant protein according to claim 1, comprising:

7. The aforementioned IL-2 mutant protein, (i) Mutation combination K35E + T37E + R38E + F42A; (ii) B'C' loop region sequences located between amino acid residues aa72 and aa84: AGDASIH, AQSKNFH or SGDASIH; and (iii) Mutations at the IL-2Rβγ binding interface selected from the group consisting of N88R, D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88Q, N88T, Q126E, and V91I, The IL-2 mutant protein according to claim 1, comprising:

8. The aforementioned IL-2 mutant protein, (i) Mutation combination K35E+T37E+R38E+F42A; and (ii) Mutation at the IL-2Rβγ binding interface selected from the group consisting of D84N, D84Q, E95N, H16N, and H16N+D84N, The IL-2 mutant protein according to claim 1, comprising:

9. The IL-2 mutant protein according to any one of claims 1 to 8, wherein the IL-2 mutant protein further comprises mutant T3A.

10. The IL-2 mutant protein according to any one of claims 1 to 9, further comprising 0 to 5 amino acid mutations compared to the wild-type human IL-2.

11. An IL-2 mutant protein according to any one of claims 1 to 9, comprising an S or A residue at the aa125 position.

12. The IL-2 mutant protein according to any one of claims 1 to 11, wherein the wild-type human IL-2 comprises the sequence shown in SEQ ID NO:

1.

13. (a) The amino acid sequence of SEQ ID NO: 516; (b) Any one amino acid sequence of sequence numbers 148, 197, 489, and 513; (c) Amino acid sequences selected from the group consisting of SEQ ID NOs: 37-147, 149-196, 198-487, 490-512, 514-515, and 517-638; or, (d) An amino acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with any one of the amino acid sequences in (a) to (c), The IL-2 mutant protein according to claim 1, comprising:

14. A fusion protein comprising the IL-2 mutant protein described in any one of claims 1 to 13, wherein the IL-2 mutant protein is fused with an Fc fragment via a linker.

15. (a) The linker is GSGS or (G4S) 2 And; (b) The Fc fragment is human IgG1 Fc; (c) The Fc fragment comprises mutation L234A + L235A according to EU numbering; (d) The Fc fragment has an amino acid sequence that is at least 95%, at least 96%, or 100% identical to SEQ ID NO: 12; and / or (e) The Fc fragment comprises the Knob mutation or mutations T366W and S354C according to EU numbering; or the Fc fragment comprises the Hole mutation or mutations Y349C, T366S, L368A and Y407V according to EU numbering; The fusion protein according to claim 14.

16. An IL-2-Fc dimer protein comprising the fusion protein described in claim 14 or 15.

17. The IL-2-Fc dimer protein according to claim 16, comprising a first monomer and a second monomer, wherein each monomer comprises, from the N-terminus to the C-terminus, i) an IL-2 mutant protein, ii) a linker, and iii) an Fc fragment.

18. The IL-2-Fc dimer protein according to claim 17, wherein the linker is (G4S)2 and the Fc fragment has the amino acid sequence of SEQ ID NO:

12.

19. The IL-2-Fc dimer protein according to claim 18, comprising an IL-2 mutant protein selected from SEQ ID NOs. 516, 148, 197, 489, and 513, wherein each monomer is linked at the C-terminus to the amino acid sequence of SEQ ID NO: 12 via a linker (G4S) 2.

20. A heterodimer comprising a first monomer and a second monomer, The first monomer comprises, from the N-terminus to the C-terminus, i) an IL-2 mutant protein, ii) a linker, and iii) a first Fc fragment; and, The second monomer comprises a second Fc fragment, The IL-2-Fc dimer protein according to claim 16.

21. The first Fc fragment and the second Fc fragment each contain first and second heterodimerizing mutations that promote the formation of the heterodimer between the first monomer and the second monomer, wherein the first heterodimerizing mutation in the first Fc fragment contains a Knob mutation and the second heterodimerizing mutation in the second Fc fragment contains a Hole mutation; or, the first heterodimerizing mutation in the first Fc fragment contains a Hole mutation and the second heterodimerizing mutation in the second Fc fragment contains a Knob mutation. The IL-2-Fc dimer protein according to claim 20.

22. The IL-2-Fc dimer protein according to claim 21, wherein the Knob mutation is T366W / S354C according to EU numbering, and the Hole mutation is Y349C / T366S / L368A / Y407V according to EU numbering.

23. The IL-2-Fc dimer protein according to any one of claims 20 to 22, wherein the linker is (G4S)2, the first Fc fragment comprises the amino acid sequence of SEQ ID NO: 9, and the second monomer comprises the amino acid sequence of SEQ ID NO:

10.

24. The IL-2-Fc dimer protein according to any one of claims 20 to 23, wherein the first monomer comprises an IL-2 mutant protein selected from SEQ ID NOs. 516, 148, 197, 489 and 513, which is linked at the C-terminus to the amino acid sequence of SEQ ID NO: 9 via a linker (G4S) 2; and the second monomer comprises the amino acid sequence of SEQ ID NO:

10.

25. An immune complex comprising an IL-2 mutant protein according to any one of claims 1 to 13 and an antigen-binding molecule, wherein the antigen-binding molecule is an antibody or an antibody fragment.

26. An isolated polynucleotide encoding an IL-2 mutant protein according to any one of claims 1 to 13, or a fusion protein according to claim 14 or 15, or an IL-2-Fc dimer protein according to any one of claims 16 to 24, or an immune complex according to claim 25.

27. An expression vector comprising the polynucleotide described in claim 26.

28. A host cell comprising the polynucleotide described in claim 26 or the vector described in claim 27.

29. A pharmaceutical composition comprising an IL-2 mutant protein according to any one of claims 1 to 13, or a fusion protein according to claim 14 or 15, or an IL-2-Fc dimer protein according to any one of claims 16 to 24, or an immune complex according to claim 25, and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition for therapeutic use comprising an IL-2 mutant protein according to any one of claims 1 to 13, or a fusion protein according to claim 14 or 15, or an IL-2-Fc dimer protein according to any one of claims 16 to 24, or an immune complex according to claim 25.

31. A pharmaceutical composition for use according to claim 30, for use in the treatment of cancer or autoimmune disease, or for use in stimulating the immune system of a subject.

32. A method for obtaining IL-2 mutant proteins, (a) A step of shortening the B'C' loop region sequence of wild-type IL-2 by mutation, and introducing one or more mutations to the IL-2Rβγ binding interface of IL-2, The shortened B'C' loop region has a sequence located between amino acid residues aa72 and aa84, and the sequence is shortened to a length of less than 10 amino acids, and The shortened B'C' loop region is, (i) The sequence of GDASIH from position aa74 to aa83; or, (ii) From aa74 to aa83, the sequence is (Q / G)S(K / A / D)N(F / I)H; Includes, The mutations at the IL-2Rβγ binding interface are as follows: The mutations include those selected from the group consisting of D20N, D84E, D84N, D84N+E95Q, D84N+Q126E, D84N+V91I, D84Q, D84T, D84T+H16T, D84T+Q126E, E15Q, E95N, E95Q, H16N, H16N+D84N, H16T, H16T+D84Q, H16T+V91I, N88D, N88R, N88Q, N88T, Q126E, and V91I; Here, the positions of the amino acids are numbered according to Sequence ID No.

1. The aforementioned step; (b) The step of expressing the IL-2 mutant protein obtained from (a) in mammalian cells in the form of fusion to Fc; and, (c) Identifying IL-2 mutant proteins having one or any combination of the following improved characteristics: (i) improved expression level and / or protein purity after one-step affinity chromatography as measured by SEC-HPLC; (ii) weakened IL-2Rβ binding and / or altered IL-2Ra binding; The method, including the method described above.

33. The method according to claim 32, wherein the mutation at the IL-2Rβγ binding interface of IL-2 includes a mutation in N88R.

34. The method according to claim 32 or 33, wherein step (a) further comprises introducing one or more mutations at the IL-2Rα binding interface of IL-2, the mutations at the IL-2Rα binding interface include a combination of K35E + T37E + R38E + F42A or a combination of K35E + T37E + R38E.

Images