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 and immunoconjugates containing the IL-2 muteins, as well as nucleic acids encoding the IL-2 muteins, vectors containing the nucleic acids, and host cells. The present invention also provides methods for preparing the IL-2 muteins, pharmaceutical compositions containing the IL-2 muteins, and therapeutic uses of the muteins.

Description

Technical Field

[0001] The present invention relates to a novel interleukin-2 (IL-2) mutant protein and its use. Specifically, the present invention relates to an IL-2 mutant protein having improved properties compared to wild-type IL-2, such as improved drug discovery potential, reduced IL-2Rα receptor binding ability, and / or increased IL-2Rβ receptor binding ability. The present invention further provides a fusion protein, an immune complex containing the IL-2 mutant protein, a nucleic acid encoding the IL-2 mutant protein, a vector containing the nucleic acid, and a host cell. The present invention further provides a method for preparing and screening the IL-2 mutant protein, a pharmaceutical composition containing the IL-2 mutant protein, and a therapeutic use of the mutant protein.

Background Art

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a pleiotropic cytokine mainly produced by activated T cells, particularly CD4+ T helper cells. In eukaryotic cells, human IL-2 (uniprot:P60568) is synthesized as a precursor polypeptide of 153 amino acids, and after removing the 20 amino acids at the N-terminus, mature secreted IL-2 is produced. Sequences of IL-2 from other species are also 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 are expressed on cytotoxic CD8+ T cells and natural killer (NK) cells, while trimer receptors are mainly expressed on activated lymphocytes and CD4+CD25+FoxP3+ inhibitory regulatory T cells (Treg) (Byman, O. and Sprent, J. Nat. Rev. Immunol. 12, 180-190 (2012)). Quiescing effector T cells and NK cells are relatively insensitive to IL-2 because they do not have CD25 on their cell surface. 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, as an immune system stimulant, IL-2 can stimulate T cell proliferation and differentiation, induce the production of cytotoxic T lymphocytes (CTLs), promote B cell proliferation and differentiation and immunoglobulin synthesis, and stimulate the production, proliferation, and activation of natural killer (NK) cells. For these reasons, it is approved for use as an immunotherapy in the treatment of cancer and chronic viral infections. On the other hand, IL-2 promotes the maintenance of immunosuppressive CD4+CD25+ 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 can mediate activation-induced cell death (AICD), contributing to the establishment and maintenance of immune tolerance to autoantigens and tumor antigens (Lenardo et al., Nature 353:858 (1991)), thereby causing AICD-induced tumor resistance and activated Treg cell-mediated immunosuppression in patients. High doses of IL-2 may cause vascular leakage syndrome (VLS) in patients. It has been shown that IL-2 induces pulmonary edema by directly binding to the IL-2 trimer receptor (IL-2αβγ) on pulmonary endothelial cells (Krieg et al., Proc Nat Acad Sci USA107,11906-11(2010)).

[0006] To overcome the aforementioned problems associated with IL-2 immunotherapy, it has been proposed to reduce the toxicity of IL-2 therapy and / or improve its efficacy by altering the selectivity or preference of IL-2 for different receptors. For example, it has been proposed to enhance the in vivo IL-2 therapeutic effect by inducing preferential amplification in the CD122-high population by targeting IL-2 to cells expressing CD122 instead of CD25 using a monoclonal antibody-IL-2 complex (Boyman et al., Science 311, 1924-1927 (2006)). Oliver AST et al. (US2018 / 0142037) have proposed introducing the triple mutation F42A / Y45A / L72G at amino acid residue positions 42, 45, and 72 of IL-2 to reduce its affinity for the IL-2Rα receptor. Aron M. Levin et al. (Nature, Vol 484, pp. 529-533, DOI: 10.1038 / nature10975) proposed an IL-2 mutant called "superkine," IL-2H9, which contains a quintuple mutation L80F / R81D / L85V / I86V / I92F and has enhanced IL-2Rβ binding, thereby increasing its stimulating effect on CD25- cells while maintaining high binding affinity to CD25. Rodrigo Vazquez-Lombardi et al. (Nature Communications, 8:15373, DOI:10.1038 / ncomms15373) proposed a triple mutant human IL-2 mutant protein, IL-23X. This protein has residue mutations R38D-K43E-E61R at amino acid residue positions 38, 43, and 61, respectively. As a result, this mutant protein does not bind to IL-2Rα, but it has a weak effect in activating CD25- cells, and a bias in activation towards CD25+ cells still exists. Rodrigo Vazquez-Lombardi et al. also proposed improving the pharmacodynamic properties of interleukins by preparing interleukin 2-Fc fusions, but the expression level of these fusion proteins is low and they tend to form aggregates.

[0007] Given the role of IL-2 in immunomodulation and disease, there remains a need in this field to develop novel IL-2 molecules with improved properties, particularly those useful in production and purification, and those with improved pharmacodynamic properties. [Overview of the project]

[0008] The present invention satisfies the above requirements by providing a novel IL-2 mutant protein having improved drug potential and / or improved IL-2 receptor selectivity / bias compared to wild-type IL-2.

[0009] 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) Reduction or elimination of binding to IL-2Rα, (iii) Enhanced binding with IL-2Rβ.

[0010] In some embodiments, the present invention provides an IL-2 mutant protein that includes a mutation at the binding interface of IL-2 to IL-2Rα and has a shortened B'C' loop sequence.

[0011] The present invention provides a fusion protein and immune complex containing an IL-2 mutant protein, a pharmaceutical composition and combination product, a nucleic acid encoding the IL-2 mutant protein, a vector and host cell containing the nucleic acid, and a method for producing the IL-mutant protein, fusion protein and immune complex of the present invention.

[0012] Furthermore, the present invention also provides methods for treating diseases using the IL-2 mutant protein, fusion, and immune complex of the present invention, as well as methods and uses for stimulating the immune system of a subject.

[0013] 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]

[0014] [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] Primers for constructing the mutation library IBYDL029 are shown. [Figure 4] This shows IL-2 mutant proteins and their sequences screened from the mutant library IBYDL029. [Figure 5A] The selected and constructed IL-2mutant-FC fusion protein shows signaling curves that activate p-STAT5 in CD8+CD25-T cells (A) and CD8+CD25+T cells (B). [Figure 5B] The selected and constructed IL-2mutant-FC fusion protein shows signaling curves that activate p-STAT5 in CD8+CD25-T cells (A) and CD8+CD25+T cells (B). [Figure 6A] The in vivo antitumor effect of the IL-2mutant-FC fusion protein Y092 (A) and the body weight changes in animals monitored after administration (B and C) are shown. [Figure 6B] The in vivo antitumor effect of the IL-2mutant-FC fusion protein Y092 (A) and the body weight changes in animals monitored after administration (B and C) are shown. [Figure 6C] The in vivo antitumor effect of IL-2mutant-FC fusion protein Y092 (A) and the body weight changes of the animals monitored after administration (B and C) are shown. [Figure 7A] The in vivo antitumor effect of IL-2mutant-FC fusion protein Y144 (A) and the body weight changes of the animals monitored after administration (B and C) are shown. [Figure 7B] The in vivo antitumor effect of IL-2mutant-FC fusion protein Y144 (A) and the body weight changes of the animals monitored after administration (B and C) are shown. [Figure 7C] The in vivo antitumor effect of IL-2mutant-FC fusion protein Y144 (A) and the body weight changes of the animals monitored after administration (B and C) are shown. [Figure 8] The amino acid sequence of wild-type IL-2 protein IL-2WT (SEQ ID NO: 1) and its amino acid residue numbering are shown, and the sequence alignment with mutant protein IL-23X is shown. [Figure 9] The entire sequence of yeast display plasmid pYDC011 is shown.

Mode for Carrying Out the Invention

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For the purposes of the present invention, the following terms are defined below.

[0016] The term "about", when used with a number or numerical value, means covering a number or numerical value within a range that is 5% less than the number or numerical value specified as the lower limit and 5% greater than the number or numerical value specified as the upper limit.

[0017] The term "and / or" should be understood to refer to any one of the selectable options, any two of the selectable options, or a combination of a plurality of the selectable options

[0018] 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.

[0019] 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 (especially conservative amino acid substitutions), and preferably have substantially the same IL-2Rα binding affinity and / or IL2Rβ binding affinity as the natural IL-2 protein. Therefore, in some embodiments, wild-type IL-2 may include 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.

[0020] In this specification, amino acid mutations may include amino acid substitutions, deletions, insertions, and additions. Any combination of substitutions, deletions, insertions, and additions can be used to obtain a final mutant protein construct having desired properties (e.g., reduced IL-2Rα binding affinity and / or improved drug potential). Amino acid deletions and insertions include deletions and insertions at amino and / or carboxyl group ends of polypeptide sequences, as well as deletions and insertions within polypeptide sequences. For example, the length of a loop region can be shortened by deleting an alanine residue at position 1 of full-length human IL-2 or by 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 entire or partial B'C' loop region sequence of wild-type IL-2 can be replaced with a different sequence, preferably to obtain a shortened B'C' loop region sequence.

[0021] In this invention, when referring to an amino acid position in an IL-2 protein or IL-2 sequence segment, it is determined by referring to the amino acid sequence SEQ ID NO: 1 of wild-type human IL-2 protein (also known as IL-2WT) (shown in Figure 8). Amino acid sequence alignment with SEQ ID NO: 1 allows for the identification of the corresponding amino acid position on other IL-2 proteins or polypeptides (including full-length sequences or cleavage fragments). Therefore, in this invention, unless otherwise specified, the amino acid positions of IL-2 proteins or polypeptides are amino acid positions 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 another IL-2 polypeptide sequence. Sequence alignment performed for amino acid position determination can be done using the Basic Local Alignment Search Tool, available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] [Table 1]

[0026] For example, the wild-type IL-2 protein may have a conserved amino acid substitution in one of sequence numbers 1-3, or may have only a conserved amino acid substitution. Furthermore, for example, the mutant IL-2 protein of the present invention may have a conserved amino acid substitution in one of the IL-2 mutant protein sequences specifically shown herein (e.g., any one of sequence numbers 22-26), or may have only a conserved amino acid substitution.

[0027] "Affinity" or "binding affinity" can be used to reflect the internal binding capacity of the interaction between members of a binding pair. The affinity of molecule X for its binding partner Y may be expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). 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.

[0028] 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.

[0029] 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 follows 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.

[0030] Various aspects of the present invention will be described in further detail in the following sections.

[0031] 1. The IL-2 mutant protein of the present invention

[0032] In one embodiment, the present invention provides a novel IL-2 mutant protein having improved drug potential and / or improved IL-2 receptor selectivity / preference.

[0033] Advantageous biological properties of the IL-2 mutant protein of the present invention

[0034] The inventors have found that the binding of IL-2 mutant proteins to IL-2Rα can be reduced or eliminated by introducing one or more specific mutations at the binding interface of IL-2 to the IL-2Rα receptor. Furthermore, the inventors have found that the expression and / or purity of IL-2 can be increased and / or affinity for IL-2Rβ can be increased by substituting the B'C' loop sequence of IL-2 itself with a shorter B'C' loop sequence derived from another interleukin cytokine such as IL-15, or by cleaving the B'C' loop sequence of IL-2 itself. The inventors have further found that by combining the mutation at the binding interface to the IL-2Rα receptor with the mutation in the shortened B'C' loop region, it is possible to provide IL-2 mutants having improved properties selected from one or more of the following: (i) improved expression and / or purity, (ii) reduced or eliminated binding to the IL-2Rα receptor, and / or (iii) enhanced binding to the IL-2Rβ receptor.

[0035] Accordingly, the present invention provides an IL-2 mutant protein having one or more of the above-described improved characteristics (i) to (iii), and in particular having the improved characteristics (i) and (ii) simultaneously.

[0036] Improved drug discovery potential

[0037] In some embodiments, the IL-2 mutant protein of the present invention has improved drug potential. For example, when expressed in mammalian cells such as H293T or CHO cells, or when expressed in an Fc fusion protein, for example, it has one or more characteristics selected from the following: (i) higher expression levels than wild-type IL-2 protein, and (ii) easier purification to higher protein purity. In some embodiments, the IL-2 mutant protein of the present invention also has storage stability. For example, after storage in PBS buffer at pH 7.4 at 40°C for 14 days, the decrease in protein purity detected by SEC-HPLC is 5%, 2%, or 1% or less, or the decrease in protein purity detected by CE-SDS is 5%, 3%, or 2% or less.

[0038] 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 evaluated by Western blotting or ELISA. In some embodiments, the IL-2 mutant protein of the present invention exhibits at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 3-fold, or 4-fold or more, or at least 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, or even more than 10-fold, increase in expression levels in mammalian cells compared to wild-type IL-2.

[0039] 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, for example, following one-step protein A affinity chromatography purification according to the IL-2 fusion protein purification method described in Example 2, the purity of the IL-2 mutant protein product of the present invention can reach 70%, 80%, or 90% or higher, preferably 92%, 93%, 94%, 95%, 98%, or 99% or higher.

[0040] Improved IL-2 receptor selectivity / preference

[0041] 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 expressed on quiescent effector cells (including CD8+ T cells and NK cells). IL-2Rα, which has 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. Therefore, without being constrained by theory, reducing or eliminating IL-2's affinity for the IL-2Rα receptor reduces the bias of IL-2 in preferentially activating CD25+ cells and reduces the IL-2-mediated immunodownregulating effect on Treg cells. Without being constrained by theory, maintaining or enhancing affinity for the IL-2Rβ receptor preserves or enhances the activating effect of IL-2 on effector cells such as CD8+ T cells and NK cells, and the resulting immunostimulatory effect of IL-2.

[0042] Therefore, in some embodiments, the IL-2 mutant protein of the present invention has improved properties compared to wild-type IL-2, selected from, for example, one or more of the following: (1) Reduction or elimination of binding affinity to the IL-2Rα receptor, (2) Enhanced binding affinity to the IL-2Rβ receptor, (3) Reduction of binding affinity to high affinity IL-2R receptor (IL-2Rαβγ), (4) Increased binding affinity to moderate affinity IL-2R receptor (IL-2Rβγ), (5) Reduced ability of CD25+ cells (especially activated CD8+ T cells and Treg cells) to activate IL-2 signaling, particularly the STAT5 phosphorylation signaling pathway. (6) This results in a decrease in the activation and proliferation of IL-2-mediated CD25+ cells (especially activated CD8+ T cells and Treg cells), (7) Reduction or elimination of the bias of IL-2 that preferentially stimulates the proliferation of Treg cells. (8) Reduction of the immune downregulating effect of Treg cells under IL-2 induction, (9) Maintaining or enhancing the activating effect on CD25- cells (especially CD25-T effector cells and NK cells), especially enhancing, (10) To result in increased activation and proliferation of IL-2-mediated effector T cells and NK cells.

[0043] In some embodiments, the IL-2 mutant protein of the present invention has the characteristics of (1) above, preferably having one or more of the characteristics selected from (3) and (5) to (8), in particular all of them, and more preferably having one or more of the characteristics selected from (2), (4) and (9) or (10), in particular all of them. In some embodiments, the IL-2 mutant protein of the present invention has the characteristics of (2) and (4) above, preferably having one or more of the characteristics selected from (9) or (10), in particular all of them, and more preferably having one or more of the characteristics selected from (1), (3) and (5) to (8), in particular all of them.

[0044] In a preferred embodiment, the IL-2 mutant protein of the present invention exhibits in vivo antitumor effects, such as an antitumor effect against colon cancer.

[0045] In some preferred embodiments, the IL-2 mutant protein of the present invention further exhibits reduced in vivo toxicity mediated by the binding of IL-2 to the high-affinity receptor IL-2αβγ, compared to wild-type IL-2.

[0046] In some preferred embodiments, the IL-2 mutant protein of the present invention does not cause significant toxicity to subjects after administration, as reflected, for example, by changes in the subject's body weight after administration. For example, compared to before administration, after administration for a certain period, for example, 20 days or more, the decrease in body weight is 15% or less, 10% or less, or 5% or less.

[0047] In some embodiments, the IL-2 mutant protein of the present invention exhibits a binding affinity to the IL-2Rα receptor that is at least 5-fold, at least 10-fold, or at least 25-fold, and particularly at least 30-fold, 50-fold, or 100-fold or more, compared to wild-type IL-2 (e.g., IL-2WT shown in SEQ ID NO: 1). In preferred embodiments, the mutant protein of the present invention does not bind to the IL-2 receptor α. Binding affinity can be measured by measuring the equilibrium dissociation constant (KD) 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, and the receptor IL-2Rα receptor using ForteBio affinity measurement technology.

[0048] In some embodiments, the IL-2 mutant protein of the present invention exhibited, for example, a 5- to 10-fold or greater enhanced binding affinity to the IL-2Rβ receptor compared to wild-type IL-2 (e.g., IL-2WT as shown in SEQ ID NO: 1). Binding affinity can be measured by ForteBio affinity measurement technology, which measures the key-to-dissociation (KD) value between the IL-2 mutant protein of the present invention, such as the IL-2 mutant protein fused with an Fc fragment, and the IL-2Rβ receptor. In one embodiment, in the form of an IL-2-Fc fusion protein, the divalent binding affinity KD value between the IL-2 mutant protein of the present invention and the IL-2Rβ receptor in ForteBio affinity measurement (e.g., ForteBio affinity measurement described in the Examples) was less than 10.0E-09M, for example, between 1.0E-09M and 7E-09M, or less than 1.0E-09M, for example, between 1.0E-10M and 7.0E-10M.

[0049] In one embodiment, compared to wild-type IL-2, the IL-2 mutant protein of the present invention results in reduced activation and / or proliferation of IL-2-mediated CD25+ cells. In one embodiment, the CD25+ cells are CD25+CD8+ T cells. In another embodiment, the CD25+ cells are Treg cells. In one embodiment, in a STAT5 phosphorylation assay, the ability of the IL-2 mutant protein to activate CD25+ cells is identified by detecting the activation of the STAT5 phosphorylation signal in CD25+ 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.

[0050] In one embodiment, compared to wild-type IL-2, the IL-2 mutant protein of the present invention results in the activation and / or proliferation of CD25-effector cells mediated by maintained or enhanced IL-2. In one embodiment, CD25-cells are CD8+ effector T cells or NK cells. In one embodiment, in a STAT5 phosphorylation assay, the ability of the IL-2 mutant protein to activate CD25-cells is identified by detecting the EC50 value of the IL-2 mutant protein that activates the STAT5 phosphorylation signal in CD25-cells. In one embodiment, as measured in a STAT5 phosphorylation assay, the IL-2 mutant protein of the present invention showed at least a 1-fold, e.g., 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold increase in the ability to activate CD25-cells compared to wild-type IL-2 protein (e.g., human IL-2 of SEQ ID NO: 1).

[0051] In one embodiment, compared to wild-type IL-2, the IL-2 mutant protein of the present invention eliminates or reduces the bias of IL-2 in preferentially activating CD25+ cells. In one embodiment, CD25+ cells are CD25+CD8+ T cells. In another embodiment, CD25+ cells are Treg cells. In one embodiment, in a STAT5 phosphorylation assay, the ability of the IL-2 mutant to activate CD25- cells is identified by detecting the EC50 values ​​of the IL-2 mutant protein that activate the STAT5 phosphorylation signal in CD25- cells and CD25+ cells, respectively. For example, the activation bias of the IL-2 mutant protein to CD25+ cells is determined by calculating the ratio of EC50 values ​​that activate the STAT5 phosphorylation signal in CD25- and CD25+ T cells. Preferably, compared to the wild-type protein, the bias of the mutant protein to CD25+ was reduced by at least 10-fold, preferably at least 100-fold, 150-fold, 200-fold, 300-fold or more.

[0052] Mutant protein of the present invention

[0053] The IL-2 protein belongs to the family of short-chain type I cytokines, possessing a structure of four α-helix bundles (A, B, C, D). Analysis of its crystal structure (PDB: 1Z92) reveals that IL-2 has amino acid sites 35, 37, 38, 41, 42, 43, 45, 61, 62, 68, and 72 in the 35-72 amino acid region that interact with CD25 (i.e., IL-2Rα). The inventors have found that the binding of IL-2 to IL-2Rα can be reduced or eliminated by introducing specific mutations into the CD25 interaction sites (i.e., sites 35, 37, 38, 41, 42, 43, 45, 61, 62, 68, and 72) in the 35-72 amino acid region of IL-2. In this specification, mutations occurring at these sites in the region are abbreviated as "CD25-binding region" mutations.

[0054] Furthermore, by comparing the crystal structures of the IL-2 monomer (PDB:1M47) and the complex (PDB:2ERJ), the inventors found that the B'C' loop is highly active in solution and cannot form a relatively stable conformation, indicating that the B'C' loop is deleted in the crystal structure of the IL-2 monomer. By improving the genetic engineering of the B'C' loop, such as through sequence substitution or cleavage, the stability of the B'C' loop can be improved, thereby improving the drug potential of IL-2 and / or its binding affinity to the IL-2Rβ receptor. In this specification, these sequence substitution or cleavage mutations occurring in the B'C' loop region are abbreviated as "B'C' loop region mutations."

[0055] The inventors have further discovered that a combination of a "CD25 binding region" mutation and a "B'C' loop region mutation" can be used to further improve the properties of IL-2. Thus, the present invention provides an IL-2 mutant protein comprising (i) a "CD25 binding region" mutation and (ii) a "B'C' loop region mutation" compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 containing sequence number 1).

[0056] Mutation in the CD25 binding region

[0057] In one embodiment, the IL-2 mutant protein of the present invention comprises one or more mutations that eliminate or reduce the binding affinity to the IL-2Rα receptor in the CD25 binding region, preferably at positions 35, 37, 38, 41, 42, 43, 45, 61, 68, and 72, compared to wild-type IL-2.

[0058] In some embodiments, the mutations in the CD25 binding region of the present invention include a combination of mutations selected from one of the following combinations (1) to (9):

[0059] [Table 2]

[0060] In a preferred embodiment, the mutations in the CD25 binding region of the present invention include a combination of mutations selected from one of the following combinations (1) to (9), particularly a combination of mutations selected from one of the following combinations (1) to (6):

[0061] [Table 3]

[0062] In one preferred embodiment, the CD25 binding region mutation of the present invention includes or consists of the mutation combination K35E+T37E+R38E+F42A.

[0063] In a preferred embodiment, the mutation in the CD25 binding region of the present invention results in eliminated CD25 binding, for example, CD25 binding below the detection limit using ForteBio affinity measurement.

[0064] For mutations in the CD25 binding region 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.

[0065] B'C' loop region mutation

[0066] 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.

[0067] 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 A74-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.

[0068] 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.

[0069] 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), where 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 SEQ ID NO: 1.

[0070] 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)S(K / A / D)N(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)S(K / A / D)N(F / I)H or substitution by GDASIH.

[0071] 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).

[0072] 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)S(K / A / D)N(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:

[0073] [Table 4]

[0074] In one preferred embodiment, the IL-2 mutant protein of the present invention comprises a B'C' loop region sequence selected from AQSKNFH, AQSANFH, AQSDNFH, or SGDASIH or AGDASIH.

[0075] 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.

[0076] Preferred exemplary mutation combinations

[0077] In some preferred embodiments, the combination of the B'C' loop mutation of the present invention and the CD25 binding region mutation of the present invention provides improved properties selected from two or all three of the following: (i) reduced (or eliminated) IL-2Rα binding, (ii) enhanced IL-2Rα binding, and (ii) improved expression levels and purity.

[0078] In some embodiments, the present invention provides IL-2 mutant proteins comprising the following compared to wild-type IL-2: (i) A combination of mutations selected from one of the following combinations (1) to (9), in particular a combination of mutations selected from one of the combinations (1) to (6):

[0079] [Table 5]

[0080] (ii) B'C' loop region array selected from the following:

[0081] [Table 6]

[0082] In particular, a B'C' loop region sequence selected from AQSKNFH, AQSANFH, AQSDNFH, SGDASIH, or AGDASIH. Preferably, the present invention provides an IL-2 mutant protein comprising the following compared to wild-type IL-2: (i) Mutation combinations K35E+T37E+R38E+F42A, (ii) B'C' loop region sequence selected from AQSKNFH, AQSANFH, AQSDNFH, SGDASIH, and AGDASIH.

[0083] Accordingly, in one embodiment, the present invention provides an IL-2 mutant protein having 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, and comprising a ligation sequence selected from AGDASIH, SGDASIH, AQSKNFH, AQSANFH, AQSDNFH, AGSKNFH, AQSANFH, and AQSANIH between amino acid positions 72 and 84, and having the mutation combination K35E+T37E+R38E+F42A.

[0084] In some preferred embodiments, the present invention provides an IL-2 mutant protein comprising an amino acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with an amino acid sequence selected from one of SEQ ID NOs: 22, 23, 24, 25, and 26. In some embodiments, the mutant protein comprises or consists of the amino acid sequences of SEQ ID NOs: 22, 23, 24, 25, and 26.

[0085] Preferably, the combination of mutations results in a bias that preferentially stimulates p-STATA5 signaling in CD25+ T cells with reduced IL-2, and also has the property of stimulating signaling in enhanced CD25- T cells.

[0086] Other mutations

[0087] Apart from the mutations in the "CD45 binding" region and the "B'C' loop region" described above, 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-described 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. Furthermore, for example, the IL-2 mutant protein of the present invention may further include substitutions at position 76, such as K76D / A, to enhance T-cell activation activity. 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.

[0088] 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, 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, excluding both the CD25 region mutation and the B'C' loop region mutation of the present invention. In one embodiment, the remaining mutations may be conservative substitutions. In one embodiment, the remaining mutations occur outside the CD25 region and the B'C' loop region.

[0089] 2. Fusion proteins and immune complexes

[0090] The present invention further provides fusion proteins 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. In one embodiment, the Fc fragment comprises a mutation that reduces or eliminates effector function, for example, an L234A / L235A mutation or L234A / L235E / G237A that reduces binding to the Fcγ receptor. Preferably, the fusion protein comprising Fc has an increased serum half-life. In one preferred embodiment, the fusion protein comprising Fc further simultaneously has effector function mediated by the reduced Fc region, for example, reduced or eliminated ADCC, ADCP, or CDC effector function.

[0091] 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 7 or has at least 90% identity with it, for example, 95%, 96%, 97%, 99%, or more.

[0092] In some embodiments, the IL-2 mutant protein is fused to the Fc region by a linker. In some embodiments, the linker can be selected to enhance the activation effect of the Fc fusion protein on CD25-T cells. In one embodiment, the linker is GSGS, more preferably 2x(G4S).

[0093] In some embodiments, the Fc fusion protein has at least 85%, at least 95%, or at least 96% identity with an amino acid sequence selected from SEQ ID NOs: 15-19. In some embodiments, the Fc fusion protein consists of the sequences of SEQ ID NOs: 15-19.

[0094] 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.

[0095] In the fusion protein and 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.

[0096] 3. Polynucleotides, vectors, and hosts

[0097] The present invention provides nucleic acids encoding any of the above-mentioned IL-2 mutant proteins, fusions, 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.

[0098] 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 pCDNA3.1 expression vector.

[0099] 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 immune complexes are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and many cells containing the vector can be grown to inoculate into large fermenters, yielding sufficient quantities of IL-2 variants or fusions or immune complexes for clinical use. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). For example, polypeptides can be produced in bacteria, especially if glycosylation is not required. After expression, polypeptides can be isolated from bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable clones or expression hosts for polypeptide-encoding vectors, and include fungal and yeast strains in which the glycosylation pathway is already "humanized," resulting in the production of polypeptides with a partially or completely human glycosylation pattern. See Gerngross, NatBiotech22, 1409-1414 (2004) and Li et al., NatBiotech24, 210-215 (2006).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).

[0100] 4. Preparation method

[0101] In a further embodiment, the present invention provides a method for preparing the IL-2 mutant protein or fusion 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 complex, under conditions suitable for the expression of the IL-2 mutant protein or fusion or complex, and optionally recovering the protein or fusion or complex from the host cells (or host cell medium).

[0102] 5.Measurement method

[0103] 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.

[0104] 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, binding to human IL-2Rα or β protein can be measured by methods known in the art, such as ELISA, Western blotting, or 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. Alternatively, the binding of the mutant protein to the receptor, including binding dynamics (e.g., KD value), can be measured using the ForteBio assay method with recombinant mutant protein-Fc fusions.

[0105] 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.

[0106] 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.

[0107] 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 may include the steps of incubating cultured NK cells with the mutant IL-2 protein or fusion or immune complex 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 is involved in JAK (Janus kinase) and STAT (signaling molecule and transcriptional activator) signaling molecules.

[0108] The interaction between IL-2 and the receptor's β and γ subunits results in 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 to the cell nucleus, where it promotes 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 or immune complex of the present invention, and the level of phosphorylated STAT5 can be measured by flow cytometry.

[0109] 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, or immune complex of the present invention. The in vivo antitumor effect of the mutant IL-2 polypeptide, fusion, 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 vivo toxicity of the mutant IL-2 polypeptide, fusion, and immune complex of the present invention can also 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). 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).

[0110] 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.

[0111] In a further embodiment, the storage stability of the mutant IL-2 protein of the present invention can also be detected by methods known in the art. In the present invention, a “stable” antibody refers to an antibody prepared in a buffer that retains an acceptable degree of physical and / or chemical stability after storage under specific conditions. In one embodiment, the buffer is PBS buffer at pH 7.4. In another embodiment, the buffer is histidine buffer at pH 6.5. In one embodiment, the stability of the antibody is detected after storage for a certain period of time, such as two weeks or more, at 40°C. The stability of the antibody can be determined by detecting the degree of decrease in the purity of the antibody after storage using SEC-HPLC or CE-SDS.

[0112] 6. Screening Methods

[0113] In a further 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.

[0114] In one embodiment, the method of the present invention is (1) A step of introducing one or more mutations at the binding interface of IL-2 to IL-2Ra by mutation, and shortening the loop region sequence of the B'C' loop region of IL-2 by mutation, Preferably, the step involves introducing a combination of the aforementioned CD25 binding region mutations and / or a chimeric or cleavage mutation of the aforementioned B'C' loop sequence. (2) In mammalian cells (e.g., HEK293 or CHO cells), the IL-2 mutant protein is expressed in the form of, for example, an Fc fusion (e.g., an FcLALA fusion), -The step of identifying mutant proteins having improved characteristics of one or more of the following: (i) expression level and / or purity of the purified protein (e.g., purity after one-step affinity chromatography by SEC-HPLC detection), (ii) reduced IL2Rα binding, and (iii) enhanced IL2Rβ binding.

[0115] 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.

[0116] In one embodiment, the method includes identifying IL-2 mutations that confer reduced (preferably eliminated) IL-2Ra binding ability compared to wild-type IL-2, before performing the combination of mutations in step (1). In one embodiment, CD25 binding affinity is reduced or eliminated by performing amino acid substitutions or combination substitutions on sites in the CD25 binding region.

[0117] 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.

[0118] In one embodiment, a mutation in the CD25 region resulting in (e.g., known) reduced IL2Ra binding is introduced into the IL-2 protein in combination with (e.g., known) cleavage and / or substitution mutations of the B'C' loop region that improve drug potential, and then characterized. In one preferred embodiment, the characterization includes, compared to wild-type IL-2, reduced IL-2Ra binding and improved drug potential (e.g., improved expression and / or purity, and / or product stability and / or homogeneity), and optionally, essentially unchanged, weakened, or enhanced IL-2Rβ binding affinity.

[0119] 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.

[0120] 7. Pharmaceutical compositions and pharmaceutical preparations

[0121] The present invention further comprises compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an IL-2 mutant protein or its fusion or immune complex, and compositions comprising polynucleotides encoding an IL-2 mutant protein or its fusion 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.

[0122] The pharmaceutically acceptable carriers applicable to the present invention may be sterile liquids such as water and oils, including petroleum and oils derived from or synthesized from animals or plants, such as peanut oil, soybean oil, mineral oil, and sesame oil. When the pharmaceutical composition is administered intravenously, water is preferred as a vector. Furthermore, aqueous saline solutions, aqueous dextrose and glycerin solutions can be used as liquid vectors, particularly in injectable solutions. Suitable pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk powder, glycerin, propylene, diol, water, and ethanol. For further information regarding the use and applications of excipients, see also "Handbook of Pharmaceutical Excipients," 5th edition, RCRowe, PJSeskey and SCOwen, Pharmaceutical Press, London, Chicago. If necessary, the compositions may further contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard vectors such as medicinal mannitol, lactose, starch, magnesium stearate, or saccharin.

[0123] A pharmaceutical formulation comprising the present invention is prepared by mixing the IL-2 mutant protein, fusion, or immune complex of the present invention having a desired purity with any one or more pharmaceutically acceptable adjuvants (Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. An exemplary lyophilized antibody formulation is described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulation comprising a histidine acetate buffer. Sustained-release formulations can also be prepared. A suitable example of a sustained-release formulation comprises a semipermeable matrix of a solid hydrophobic polymer containing a protein, wherein the matrix is ​​a molded article, such as in the form of a film or microcapsules.

[0124] In one embodiment, the pharmaceutical composition of the present invention comprises a buffer with a pH of 6 to 8, such as PBS buffer or histidine buffer. In one embodiment, the PBS buffer is, for example, a PBS buffer with a pH of about 7.4. In one embodiment, the histidine buffer is, for example, a histidine buffer with a pH of about 6.5, comprising 10 mM histidine, 5% sorbitol, and 0.02% polysorbate 80. Preferably, the pharmaceutical composition of the present invention maintains its storage stability in the buffer.

[0125] The pharmaceutical composition or formulation of the present invention may further contain one or more other active ingredients, which are necessary for the treatment of a specific indication and preferably have complementary activities that do not adversely affect each other. Ideally, other anticancer active ingredients such as chemotherapeutic agents and immune checkpoint inhibitors should be provided. The active ingredients are present in appropriate combinations in amounts effective for the intended purpose.

[0126] 8. Combination Products

[0127] In one embodiment, the present invention further provides a combination product comprising the mutant protein or its fusion or immune complex of the present invention, 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.

[0128] 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.

[0129] 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.

[0130] 9. Treatment methods and use

[0131] 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.

[0132] 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.

[0133] 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 immune complex of the present invention. The IL-2 mutant protein of the present invention has high activity and selectivity for CD25-CD122+ effector cells (cytotoxic CD8+ T cells and NK cells) and has reduced stimulating effect on CD25+ Treg cells. Therefore, the IL-2 mutant protein of the present invention can be used in low doses to stimulate the immune system of a subject.

[0134] 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.

[0135] In another embodiment, the present invention relates to a method for treating a disease in a subject, such as cancer, comprising the step of administering to the subject an effective amount of any IL-2 mutant protein, or a fusion or immune complex thereof, as described herein. The cancer may be early, mid, or late stage cancer or metastatic cancer. In some embodiments, the cancer may be a gastrointestinal cancer, such as rectal cancer, colon cancer, or colorectal cancer.

[0136] In another embodiment, the present invention relates to a method for treating an infectious disease in a subject, such as a chronic infection, comprising the step of administering to the subject an effective amount of any IL-2 mutant protein or fragment thereof as described herein, or an immune complex, multispecific antibody, or pharmaceutical composition containing the antibody or fragment thereof. In one embodiment, the infection is a viral infection.

[0137] The mutant protein of the present invention (and pharmaceutical compositions containing the same or their fusions or immune complexes, and any other therapeutic agents) 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, depending to some extent on whether the administration is short-term or long-term, and can be administered by any suitable route, including injection, such as intravenous or subcutaneous injection. In this specification, however, various administration schedules include single doses or multiple doses at multiple times, bolus administration, pulse infusion, etc.

[0138] 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.

[0139] In a further embodiment, the present invention also provides the use of the IL-2 mutant proteins, compositions, immune complexes, and fusions of the present invention in the preparation of drugs used in the above-described methods (for example, for therapeutic purposes).

[0140] 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]

[0141] Example 1: Design and construction of an interleukin two-point mutation library

[0142] Design of an interleukin two-point mutation library

[0143] 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. The original amino acids at each site accounted for 50% of the total, and the remaining 50% was evenly divided among the "mutated amino acids" in Table 1. The theoretical diversity of the library designed for the IL-2 and IL-2Rα interaction site is 3×8×8×9×6×6×3×6×6×5×6 ≈ 2.0×10⁸, and the library was named IBYDL029 (Innoventbio Yeast Display Library).

[0144] [Table 7]

[0145] Construction of an Interleukin Two-Point Mutation Library

[0146] Wild-type IL-2 (uniprot:P60568,aa21-153,C125S, abbreviated as IL-2WT) was placed between two BamHI enzyme cleavage sites in the yeast display plasmid pYDC011 (the complete plasmid sequence is shown in Figure 9). The sequence of IL-2WT is shown in this application as Sequence ID No. 1, in which the C125S mutation is introduced at position 125 to avoid the formation of a disulfide-bridged IL-2 dimer. The specific steps for plasmid construction are as follows:

[0147] 1. The IL-2WT gene was amplified using primers AMP0210 and AMP0211 as a template (primer sequences are shown in Figure 3).

[0148] 2. Plasmid pYDC011 was enzymatically cleaved with BamHI (New England Biolab, product number: R3136V) and then recovered on a gel (QIAGEN Gel Extraction Kit, Cat.28704).

[0149] The amplification product and the enzyme cleavage product were recovered using a 3.1% agarose gel.

[0150] 4. After collection, in vitro homologous recombination was performed using the One Step Cloning Kit (Vazyme product number: C113-02).

[0151] 5. After recombination, the product was transferred to E. coli Top10 competent cells (Tenkon, product number: CB104-02), spread onto LB plates containing ampicillin resistance, and incubated overnight at 37°C.

[0152] 6. After sequencing the grown monoclonal colony, the correct plasmid was named pYDC035.

[0153] According to existing literature, the IL-2 mutant IL-23X does not bind to IL-2Rα, but maintains a constant binding affinity to IL-2Rβ (Rodrigo Vazquez-Lombardi et al, Nature Communications, 8:15373, DOI:10.1038 / ncomms15373). IL-23X was displayed on the surface of yeast and used as a control. The sequence of IL-23X is shown in SEQ ID NO: 4, and this protein, like IL-2WT, also contains the C125S mutation.

[0154] The desired primers (shown in Figure 3) were designed according to the library construction protocol in Table 1 and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0155] IBYDL029 library DNA amplification: 1. Using pYDC035 as a template, fragment 029-F was amplified using primers AMP0191 and AMP0200. 2. Using pYDC035 as a template, fragment 029-R was amplified using primers AMP0201 and AMP0199. 3. Fragments 029-F and 029-R were recovered on a gel and used as PCR amplification templates, and full-length fragment 029 was amplified using primers AMP0191 and AMP0199.

[0156] 100 μg of plasmid pYDC011 was enzymatically cleaved with BamHI, and the PCR product was recovered using a PCR product recovery kit (QIAGEN PCR Purification Kit, Cat.28104) to obtain a sufficient amount of linearized plasmid. The linearized plasmid and library DNA were mixed in a 4 μg:12 μg ratio, and the mixture of library DNA and linearized plasmid was electrically introduced into the EBY100 yeast strain using an existing method (Lorenzo Benatuil et al., An improved yeast transformation method for the generation of very large human antibody libraries. Protein Engineering, Design & Selection vol.23 no.4 pp.155-159, 2010). After electrically introducing the plasmid, the library was gradient diluted and plated on SD-Trp (TAKARA, product number: 630309), and the number of grown colonies was statistically recorded. The actual diversity of the obtained library was IBYDL029: 4.2 × 10⁸, which was greater than the theoretical diversity of the library.

[0157] Example 2: Screening and identification of an IL-2 point mutation library

[0158] Preparation and biotin labeling of IL-2Rα and IL-2Rβ proteins

[0159] Construction and transfection of expression plasmids

[0160] The IL-2 receptors IL-2Rα (Uniprot:P01589,aa22-217) and IL-2Rβ (Uniprot:P14784,aa27-240) were each constructed in a pTT5 vector (Addgene) with an avi tag (GLNDIFEAQKIEWHE, this tag peptide can be biotinylated by the BirA enzyme) and six histidine tags (HHHHHH) ligated to the C-terminus of their sequences. These vectors were then used to express IL-2Rα and IL-2Rβ proteins. The sequences of the receptors produced by these constructions are shown in SEQ ID NOs. 5 and 6.

[0161] Using polyethyleneimine (PEI, Polysciences, product number: 23966), a chemical transfection reagent, cultured HEK293-F (Invitrogen, product number: R79007) cells were transiently transfected with the expression plasmid vector constructed above, according to the protocol provided by the manufacturer.

[0162] Protein expression and purification

[0163] Cell cultures expressing IL-2Rα and IL-2Rβ proteins were centrifuged at 4500 rpm for 30 minutes, and the cells were discarded. The supernatant was filtered through a 0.22 μm filter for further purification. In short, 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 the column was washed with ten times the column volume of washing buffer (20 mM Tris pH 7.4, 300 mM NaCl, 10 mM imidazole) to remove nonspecifically binding heteroproteins. The target protein was then 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).

[0164] Biotinylation labeling of IL-2Rα and IL-2Rβ proteins

[0165] The biotin labeling method for IL-2Rα and IL-2Rβ proteins using enzyme catalysis is as follows: To an appropriate amount of IL-2Rα and IL-2Rβ protein solution 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 verified using a Fortebio Streptavidin (SA) sensor (PALL, 18-5019) to confirm the success of the biotin labeling. The biotin-labeled IL-2Rα and IL-2Rβ IH proteins obtained in this example are abbreviated as IL-2Rα-Biotin and IL-2Rβ IH-Biotin, respectively.

[0166] Screening and staining identification of IL-2 mutants using an IL-2 mutant library.

[0167] Screening for IL-2 mutants that do not bind to IL-2Rα but bind to IL-2Rβ.

[0168] From the yeast-based IL-2 mutant display library IBYDL029, 2.0 × 10⁹ yeast cells were taken, cultured, and induced, resulting in a library diversity of 2.0 × 10⁸. Due to the relatively high diversity of the IBYDL029 library, magnetic bead cell sorting was performed using Miltenyi's MACS system in the first screening. First, 2 × 10⁹ yeast cells were incubated at room temperature for 30 minutes in FACS washing buffer (1 × PBS, containing 1% bovine serum albumin), and the buffer contained 500 nM of biotin-labeled IL-2Rβ (Acro Biosystems, EZ-Link Sulfo-NHS-LC-Biotin, abbreviated as IL-2Rβ-Biotin). The cells were washed once with 50 mL of pre-cooled FACS wash buffer, then resuspended in 10 mL of the same wash buffer, and 40 μl of streptavidin microbeads (Miltenyi biotec, product number: 130-090-485) were added and incubated at 4°C for 15 min. After centrifugation at 3000 rpm for 3 min and discarding the supernatant, the cells were resuspended in 10 mL of FACS wash buffer and the cell solution was added to the Miltenyi LS column. After addition was complete, the column was washed three times with FACS wash buffer, using 3 mL of FACS wash buffer each time. The Miltenyi LS column was removed from the magnetic field, eluted with 5 mL of growth medium, and the eluted yeast cells were collected and grown overnight at 30°C.

[0169] After the first screening, the library cells obtained were shaken for 24 hours at 20°C to display IL-2 mutant, and a second sorting was performed using flow cytometry. Briefly, 3 × 10⁷ yeast cells from the library were washed three times with FACS buffer, added to FACS buffer containing IL-2Rβ-Biotin (300 nM) and Anti Flag antibody, and incubated at room temperature for 30 minutes. After washing the cells twice with FACS wash buffer, the cells were mixed with FACS wash buffer containing SA-PE (phycoerythrin-labeled streptavidin, eBioscience, product number: 12-4317-87) and goat anti-mouse IgG-conjugated Alex Flour-647 (Thermo Fisher, product number: A21235), and incubated at 4°C in the dark for 15 minutes. After washing twice with pre-cooled FACS wash buffer, the cells were resuspended in 1 mL of buffer and transferred to a filtered isolation tube. Cells were sorted using MoFlo_XDP, and the sorted yeast cells were grown overnight at 30°C. The third sorting protocol was the same as the second, and after three screenings, monoclonal cells were selected and sequenced.

[0170] Using IL-2Rβ-Biotin, 53 mutant sequences were obtained from the library IBYDL029 through three screenings.

[0171] Identification of IL-2 mutant staining

[0172] Yeast cells containing the single mutant sequence after sequencing were induced by shaking at 20°C for 24 hours to display the IL-2 mutant, and then stained with its receptors, IL-2Rα-Biotin and IL-2Rβ-IH-Biotin, respectively. The specific steps are as follows:

[0173] I. Yeast cells displayed with IL-2mutant and IL-2Rα-Biotin staining analysis:

[0174] 1. 1 × 10⁶ cells from each sample were centrifuged, the supernatant was discarded, and the cells were washed once with FACS buffer before being prepared for use.

[0175] 2. 100 μL of FACS buffer containing 50 nM IL-2Rα-Biotin and Anti-Flag antibody was added and incubated at room temperature for 30 minutes.

[0176] 3. The samples were washed twice using pre-cooled FACS buffer by centrifuging at 3000 rpm at 4°C for 3 minutes.

[0177] 4. Add 100 μL of FACS buffer containing SA-PE and goat anti-mouse conjugated Alex Flour-647, and incubate on ice in the dark for 20 minutes.

[0178] 5. After washing twice with pre-cooled FACS buffer, the cells were resuspended in 100 μL of buffer, and the binding levels of IL-2mutant and IL-2Rα were analyzed using a flow analyzer (BD, ACCURI C6).

[0179] II. Yeast cells displayed with IL-2mutant and IL-2Rβ IH-Biotin staining analysis:

[0180] 1. 1 × 10⁶ cells from each sample were centrifuged, the supernatant was discarded, and the cells were washed once with FACS buffer before being prepared for use.

[0181] 2. 100 μL of FACS buffer containing IL-2Rβ IH-Biotin (30 nM~100 nM) and an Anti-Flag antibody was added and incubated at room temperature for 30 minutes.

[0182] 3. The binding levels of IL-2mutant and IL-2Rβ were analyzed in the same manner as in steps 3-5.

[0183] As can be seen from the flow staining results, the average fluorescence signal intensity of the 53 IL-2 mutants screened from library IBYDL029 that bound to IL-2Rα was close to that of IL-23X, meaning none of them bound, while the average fluorescence signal intensity that bound to IL-2Rβ was stronger than that of IL-23X.

[0184] Measurement of IL-2mutant-FC fusion protein expression and receptor affinity (avidity).

[0185] Construction of expression plasmids

[0186] To express the IL-2mutant-FC fusion protein, the IL-2mutant sequence was ligated to FcLALA via two GGGGS sequences and constructed in a pCDNA3.1(Addgene) vector.

[0187] As a control, to express IL-2WT-FC and IL-23X-FC fusion proteins, the IL-2WT and IL-23X gene sequences were ligated to FcLALA via two GGGGS and constructed on pCDNA3.1. The Fc used in this example and subsequent examples refers to the Fc (abbreviated as FcLALA, SEQ ID NO: 7) of human IgG1 having mutations L234A and L235A.

[0188] Expression and purification of IL-2 and FC fusion proteins

[0189] A vector containing the gene encoding the above 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 under conditions of 37°C and 8% CO2, VPA was added until the final concentration reached 2 mM and 2% (v / v) feed, and the cells were cultured for another 6 days.

[0190] 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 (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 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 4 shows the sequences of the IL-2 variants of the present invention listed in Table 2.

[0191] [Table 8]

[0192] Affinity measurement between IL-2mutant-FC and its receptor

[0193] The equilibrium dissociation constant (KD) of the IL-2mutant-FC and its receptor according to the present invention was measured using the biolayer interference (ForteBio) measurement method.

[0194] 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). Briefly, the affinity of candidate IL-2mutant-FC to IL-2Rα and IL-2Rβ was measured as follows: The sensor was equilibrated offline in analytical buffer for 20 minutes, then detected online for 120 seconds to establish a baseline. Human biotinylated labeled IL-2Rα or IL-2Rβ was loaded onto an SA sensor (PALL, 18-5019) and ForteBio affinity measurements were performed. After placing the sensor loaded with IL-2Rα-Biotin or IL-2Rβ IH-Biotin in a solution containing 100 nM IL-2mutant-FC until a plateau was reached, the sensor was transferred to analytical buffer and dissociated for at least 2 minutes to measure the rate of binding and dissociation. Dynamical analysis was performed using a 1:1 coupling model.

[0195] Table 3 shows the affinity KD values ​​of IL-2mutant-FC expressed in HEK293-F and its receptor in experiments performed using the above measurement method. For control, Table 3 also shows the affinity KD values ​​of IL-2WT-FC and IL-23X-FC fusion proteins measured by the same method.

[0196] [Table 9]

[0197] As can be seen from the affinity data, all of the above mutants obtained from the IBYDL029 library block binding to IL-2Rα while simultaneously maintaining binding to IL-2Rβ.

[0198] Example 3: Construction, screening, and identification of IL-2 chimeras and cleavage mutants

[0199] Design of IL-2 B'C'loop Chimera and Severed Body

[0200] 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.

[0201] 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.

[0202] By genetically modifying the B'C'loop, we can improve its stability, thereby enhancing the stability of IL-2 and its affinity for IL-2Rβ. Therefore, we aligned 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).

[0203] [Table 10]

[0204] Construction of expression plasmids

[0205] Wild-type IL-2 (uniprot:P60568,aa21-153,C125S, abbreviated as IL-2WT), IL-2 mutant IL-23X (R38D, K43E, E61R), B'C'loop chimeric and cleaved forms were ligated to human IgG1 Fc (L234A, L235A, abbreviated as FcLALA, SEQ ID NO: 7) via GSGS ligation sequences, and constructed in a pTT5 vector, expressing the following proteins:

[0206] [Table 11]

[0207] B'C'loop chimeric forms (Y017) or cleaved forms (Y057) were screened from a library and combined with mutant Y30E1 (K35E, T37E, R38E, F42A) to ligate to FcLALA via two GGGGS and constructed in a pCDNA3.1 vector to express the following proteins. Here, Y092 has the chimeric B'C'loop loop sequence AGDASIH and removes the potential N-glycosylation caused by amino acid residues NLS at positions 71-73 of Y017. Y093 and Y094 further introduced amino acid substitutions K76A or K76D into the cleaved loop sequence based on Y089 to enhance the T-cell activation activity of the B'C'loop cleaved form. Y144 increased T3A based on Y092 to remove the N-terminal O-glycosylation of IL2.

[0208] [Table 12]

[0209] Furthermore, IL-2WT and IL-23X were linked to FcLALA via two GGGGS molecules and constructed in a pCDNA3.1 vector, and the following proteins were expressed.

[0210] [Table 13]

[0211] The specific sequence information for the above protein molecules is shown in the sequence listing.

[0212] Expression and purification of IL-2 fusion protein

[0213] 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 2, which is used for the expression of the IL-2-Fc fusion protein. Expression in CHO cells was performed as follows.

[0214] ExpiCHO cells (Invitrogen) were passaged according to the required cell volume, and the cell density was adjusted to 3.5 × 10⁶ cells / mL the day before transfection. Cell density (approximately 8–10 × 10⁶ cells / mL) was detected on the day of transfection, and the viability reached over 95%. TM Cell density was adjusted to 6 × 10⁶ cells / mL using Expression Medium (Gibco product number: A29100-01). 8% (v / v) OptiPRO™ SFM (Gibco product number: 12309-019) of the final volume was used as the transfection buffer. A corresponding amount of plasmid (0.8 μg / mL cells) was added, and the mixture was homogeneously mixed. The mixture was then filtered through a 0.22 μm filter membrane to remove bacteria, and the reagent from ExpiFectamine™ CHO Transfection Kit (Gibco, product number: A29130) was added at a cell ratio of 3.2 μL / mL. 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 cultured for another 6 days.

[0215] 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).

[0216] 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.

[0217] [Table 14]

[0218] Fusion proteins containing the B'C'loop chimeric form (Y092 / 144), cleaved forms (Y089 / 093 / 094), 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.

[0219] [Table 15]

[0220] Affinity measurement of IL-2 mutant Fc fusion proteins and their receptors

[0221] The affinity KD values ​​of the following mutant proteins were measured according to the ForteBio affinity measurement method described in Example 2. The results are shown in Table 7 below.

[0222] [Table 16]

[0223] 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 not only increased the expression level and purity of the molecule but also further increased the affinity between the molecule and IL-2Rβ. 3) The combination molecule of Y30E1 and the B'C'loop mutant blocked IL-2Rα, increased the expression level and purity of the molecule, and simultaneously improved its binding activity to IL-2Rβ.

[0224] Example 4: In vitro functional experiment of IL-2 mutant

[0225] IL-2WT has a higher affinity for IL-2Rα than IL-2Rβ and IL-2Rγ, preferentially binding to IL-2Rα on the cell surface, recruiting IL-2Rβγ, and releasing downstream p-STAT5 signaling via IL-2Rβγ, stimulating the proliferation of T cells and NK cells. Because IL-2Rα is present on the surface of Treg cells but not on the surface of effector T cells and NK cells, under normal circumstances IL-2WT preferentially stimulates the proliferation of Treg cells, downregulating the immune response. IL-2mutant does not bind to IL-2Rα, eliminating the preference for preferential stimulation of Treg cell proliferation, while simultaneously stimulating the proliferation of T cells and NK cells, effectively increasing the number of effector T cells and NK cells, and enhancing the antitumor effect.

[0226] This example investigated the removal of the activation bias of each mutant towards CD25+ cells by detecting the activation of p-STAT5 signaling in primary human CD8+ T cells by each IL-2 mutant-FC, and screened for mutants that exhibited strong activation effects towards CD25- cells. The specific steps are as follows:

[0227] 1. Resuscitation of PBMC cells:

[0228] 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.

[0229] 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.

[0230] c) The cells were resuspended in 20 mL of culture medium and incubated overnight in a 37°C carbon dioxide incubator.

[0231] 2. Purification of human CD8+ T cells:

[0232] a) The cell suspension from Step 1 was aspirated, centrifuged, and the supernatant was discarded.

[0233] b) The antibody mixture was added to 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 then negatively screened and the cells were resuspended.

[0234] c) After homogeneous mixing, the mixture was incubated at 4°C for 20 minutes, shaking once every 5 minutes.

[0235] d) After incubation, 10 mL of Robosep buffer was added, and the samples were centrifuged and washed twice.

[0236] e) Simultaneously, 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 minute, and the supernatant was discarded to pre-wash the magnetic microspheres.

[0237] f) Each sample was resuspended in 1 mL of Robosep buffer, and after being uniformly mixed, it was rotated and incubated at room temperature for 30 minutes.

[0238] g) After incubation, 6 mL of Robosep buffer was added, the mixture was placed on a magnetic stand for 1 minute, and the supernatant was collected.

[0239] h) The collected liquid was placed back on the magnetic stand for 1 minute, and the supernatant was collected.

[0240] i) The solution was centrifuged, the supernatant was discarded, and the mixture was resuspended in preheated T medium to adjust the density to 1 × 10⁶ / mL.

[0241] j) One-third of the cells were collected and, if necessary, CD25 expression was stimulated. The remaining cells were placed in a 37°C carbon dioxide incubator and cultured overnight.

[0242] 3. CD8+ T cells were stimulated to express CD25:

[0243] a) Magnetic microspheres containing anti-human CD3 / CD28 antibody (GIBCO product number: 11131D) were added to 1 / 3 of the CD8+ T cells purified in Step 2, resulting in a cell-to-microsphere ratio of 3:1.

[0244] b) The samples were placed in a carbon dioxide incubator at 37°C and left to stand for 3 days.

[0245] c) Add 10 mL of culture medium and wash twice.

[0246] d) Add culture medium and adjust to a cell density of 1 × 10⁶ / mL, then place in a 37°C carbon dioxide incubator and culture for 2 days.

[0247] 4. Detection of cell purity and expression levels:

[0248] a) CD8 and CD25 in cells were detected using anti-human CD8-PE (Invitrogen product number: 12-0086-42), anti-human CD25-PE (eBioscience product number: 12-0259-42), and isotype control antibody (BD product number: 556653).

[0249] b) The cells in Step 2 were CD8+CD25-T cells, and the cells in Step 3 were CD8+CD25+T cells.

[0250] 5. Detection of EC50 of p-STAT5 signaling activation in CD8+CD25-T cells by each IL-2mutant-FC:

[0251] a) CD8+CD25-T cells were placed in a 96-well U-bottom culture plate (Costar product number: CLS3799-50EA) at a rate of 1 × 10⁵ cells per well.

[0252] b) 100 μL each of IL-2mutant-FC, commercial IL-2 (R&D product number: 202-IL-500), IL-2WT-FC, and IL-23X-FC were added, and a 4-fold gradient dilution was performed starting from a maximum concentration of 266.7 nM, resulting in a total of 12 gradients. These were incubated in a 37°C incubator for 20 minutes.

[0253] c) 55.5 μL of 4.2% formaldehyde solution was added, and the sample was fixed at room temperature for 10 minutes.

[0254] d) Centrifuge to discard the supernatant, add 200 μL of ice-cold methanol (Fisher product number: A452-4), resuspend the cells, and incubate at 4°C in a refrigerator for 30 min.

[0255] e) Centrifuge the supernatant and wash three times with 200 μL of staining buffer (BD product number: 554657).

[0256] f) Add 200 μL of permeabilization / fixation buffer (BD product number: 51-2091KZ) containing anti-p-STAT5-AlexFlour647 (BD product number: 562076, diluted 1:200), and incubate for 3 h at room temperature in the dark.

[0257] g) Wash three times with staining buffer, resuspend the cells in 100 μL of staining buffer, and perform flow cytometry detection.

[0258] h) Using the concentration of the IL-2 molecule on the x-axis and the median fluorescence value of AlexFlour647 on the y-axis, create the EC50 value of the p-STAT5 signal. The results are shown in Figure 5A and Table 8.

[0259] 6. Detection of the EC50 of p-STAT5 signal activation in CD8+CD25+ T cells by each IL-2mutant-FC:

[0260] a) Seed CD8+CD25+ T cells at 1×105 cells per well in a 96-well U-bottom culture plate.

[0261] b) Similar to b-h in Step 5, create the EC50 value of the p-STAT5 signal. The results are shown in Figure 5B and Table 8.

[0262]

Table 17

[0263] As is evident from the experimental results, 1) the Y30E1 B'C'loop mutants Y089, Y092, Y093, and Y094 all had lower EC50 values ​​than Y30E1, which activates the p-STAT5 signal in CD25-CD8+ T cells, indicating that the optimized B'C'loop can enhance molecular activation in CD25-CD8+ T cells, which is consistent with the IL-2Rβ affinity data. 2) From the CD25-EC50 / CD25+EC50 ratio results, the mutant proteins Y30E1, Y089, Y092, Y093, and Y094 significantly reduced their bias toward CD25+ cell activation compared to Y045 (IL-2WT-Fc), indicating that the Y30E1 mutation safely blocks binding to CD25.

[0264] Example 5: Stability of IL-2 mutants

[0265] Y089, Y092, and Y094 were stored in PBS buffer (pH 7.4, Gibco, product number: 10010-023) or histidine buffer (10 mM histidine, 5% sorbitol, 0.02% polysorbate 80, adjusted to pH 6.5 with hydrochloric acid), and their stability was evaluated after storage in a 40°C constant temperature incubator (Shisei SHP-150) for 7 and 14 days, respectively. The detection indicator was the purity of the IL-2 mutant protein, measured by SEC-HPLC and CE-SDS, respectively.

[0266] [Table 18]

[0267] [Table 19]

[0268] The results showed that Y089, Y092, and Y094 all exhibited excellent stability after being left in PBS buffer and histidine buffer for 14 days.

[0269] Example 6: In vivo antitumor effect of IL-2 mutant molecule

[0270] To demonstrate the in vivo efficacy of the IL-2 mutant molecule, CT26 cells (mouse colon cancer cell line, ATCC) were inoculated into Balb / c mice, and the antitumor effect of the IL-2 mutant molecule (Y092) of the present invention was measured. SPF-grade female Balb / c mice (18-20g, purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used for the experiment, and the conformity certificate number was NO.1811230011.

[0271] CT26 cells were regularly subcultured and used for subsequent in vivo experiments. Cells were collected by centrifugation, and CT26 cells were dispersed in PBS (1×) to prepare a cell suspension with a cell concentration of 2.5 × 10⁶ cells / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank of Balb / c mice to establish a CT26 tumor-bearing mouse model.

[0272] 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 10.

[0273] [Table 20]

[0274] *:h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.

[0275] The use concentrations of h-IgG and Y092 were 10 mg / mL and 6.4 mg / mL respectively, and they were administered a total of 2 times every 7 days (Q7D×2). They were administered on the 7th and 14th days after CT26 cell inoculation respectively. As shown in Figures 6A, 6B and 6C, the tumor volume and body weight of the mice were monitored 2 - 3 times a week, and the monitoring was terminated 21 days later. On the 21st day after inoculation, the relative tumor inhibition rate (TGI%) was calculated. The calculation formula was TGI% = 100%×(tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Measurement of tumor volume: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using calipers, and the tumor volume was calculated according to the following formula: V = L×W2 / 2. The body weight was measured using an electronic balance.

[0276] The results of the tumor inhibition rate are shown in Table 11. On the 21st day after inoculation, compared with h-IgG, the single-agent inhibition rates of Y092, 0.004 mg / kg, Y092, 0.02 mg / kg, Y092, 0.1 mg / kg, and Y092, 0.5 mg / kg were 3.7%, 16.2%, 43.8%, and 53.5% respectively. From the results, it was shown that the improved IL2 molecule (Y092) had an anti-tumor effect and a dose-effect. At the same time, from the detection results of the mouse body weight (Figures 6B - 6C), it was shown that from the start of inoculation to 21 days, there was a body weight drop exceeding 10% in the high-dose group (Y092, 0.5 mg / kg), and the body weight drop of the mice in other groups did not exceed 5%. None of the mice in each administration group died.

[0277]

Table 21

[0278] Example 7: In Vivo Anti-Tumor Effect of IL-2 Variant Molecules

[0279] Furthermore, the molecule Y092 was mutated (T3A) to reduce its glycosylation. To demonstrate the in vivo efficacy of the IL-2 mutant molecule, MC38 cells (mouse colon cancer cell line, ATCC) were used to inoculate C57 mice, and the antitumor effect of the IL-2 mutant molecule of the present invention (Y144) was measured. SPF-grade female C57 mice (15-18g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used for the experiment, and the certificate of conformity number was NO.1100111911070497.

[0280] MC38 cells were regularly subcultured and used for subsequent in vivo experiments. Cells were collected by centrifugation, and MC38 cells were resuspended in PBS (1×) to prepare a cell suspension with a cell concentration of 5 × 10⁶ 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.

[0281] 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 12.

[0282] [Table 22]

[0283] *:h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.

[0284] The concentrations used for h-IgG and Y144 were 10 mg / mL and 0.5 mg / mL, respectively, and were administered a total of three times every 7 days (Q7D × 3). Administration was performed on days 7, 14, and 21 after MC38 cell inoculation, and the tumor volume and body weight of mice were monitored twice a week, as shown in Figures 7A, 7B, and 7C, until monitoring was discontinued after 24 days. The relative tumor suppression rate (TGI%) was calculated on day 24 after inoculation. The formula was TGI% = 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-administration 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 according to the following formula: V = L × W² / 2. Body weight was measured using an electronic balance.

[0285] The tumor suppression rates are shown in Table 13. On day 24 after vaccination, the monotherapy suppression rate of Y144 was 30.05% compared to the h-IgG 1 mg / kg group. Simultaneously, mouse body weight detection results (Figure 7C) showed no significant difference in mouse body weight on day 24 after vaccination.

[0286] Explanation of the sequence list

[0287] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5]

Claims

1. Compared to wild-type IL-2, this IL-2 mutant protein contains a shortened B'C' loop region, With respect to the B'C' loop region of wild-type human IL-2, which consists of amino acid residues aa73 to aa83, the shortened B'C' loop region is as follows: (i) In wild-type IL-2, the sequence from position (aa)73 to (aa)83 in the B'C' loop region is replaced with the sequence of AGDASIH or SGDASIH; (ii) The sequence from position (aa)73 to (aa)83 of the B'C' loop region of wild-type IL-2 is replaced with AQSKNFH, AQSANFH, AQSDNFH, AGSKNFH, or AQSANIH; or, (iii) The sequence at positions aa73 to aa83 of the B'C' loop region of wild-type IL-2 has been cleaved by 4 amino acids from the C-terminus; Having either of the following, Here, the positions of the amino acids are numbered according to Sequence ID No.

1. The aforementioned IL-2 mutant protein.

2. The IL-2 mutant protein according to claim 1, further comprising a mutation at the binding interface of IL-2 to CD25 compared to wild-type IL-2.

3. The mutation at the binding interface between IL-2 and CD25 is, (a) K35E+T37E+R38E+F42A; (b) K35E+T37D+R38W+F42Q+Y45K+E61K+E68R; (c) K35D+R38E+T41E+K43E; (d) K35D+T37E+R38D+K43Y+Y45K+L72F; (e) K35E+R38E+T41E+K43Y+Y45K+L72F; (f) K35D+T37E+R38D+T41E+K43E+L72F; (g) K35D+T37E+R38D+K43E+L72F; (h) K35E + T37D + R38D + K43E + L72F; and (i) K35E+R38D+T41E+K43E+E61K+L72F; A mutant protein according to claim 2, selected from the group consisting of the following.

4. The mutant protein according to claim 1, further comprising K35E + T37E + R38E + F42A.

5. The mutant protein according to any one of claims 1 to 4, wherein the shortened B'C' loop region consists of a sequence selected from the group consisting of AGDASIH, AQSKNFH, AQSANFH, AQSDNFH, AGSKNFH, and AQSANIH.

6. The mutant protein according to any one of claims 1 to 4, wherein the shortened B'C' loop region consists of the sequence AGDASIH.

7. Compared to wild-type IL-2, (i) K35E + T37E + R38E + F42A, and (ii) The shortened B'C' loop region, consisting of a sequence selected from the group consisting of AGDASIH, AQSKNFH, AQSANFH, AQSDNFH, and SGDASIH, The mutant protein according to claim 1, comprising:

8. The mutant protein according to any one of claims 1 to 7, wherein the mutant protein further comprises mutant T3A.

9. The mutant protein according to any one of claims 1 to 8, wherein the mutant protein further comprises 0 to 5 additional amino acid mutations compared to wild-type IL-2.

10. The mutant protein according to any one of claims 1 to 9, wherein the wild-type IL-2 comprises human-derived IL-2 or the sequence of SEQ ID NO: 1, 2, or 3.

11. The aforementioned mutant protein is as follows: (i) an amino acid sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 22, 24, 25, and 26; or (ii) Amino acid sequence of sequence numbers 23, 22, 24, 25, or 26, A mutant protein according to any one of claims 1 to 10, comprising:

12. The mutant protein according to any one of claims 1 to 11, which, when expressed in mammalian cells as an Fc fusion protein, has improved expression levels and / or higher purity, as measured by SEC-HPLC detection after one-step affinity chromatography, compared to wild-type IL-2.

13. Compared to wild-type IL-2, it possesses one or more of the following characteristics: - Reduces binding affinity to high-affinity IL-2R receptor (IL-2Rαβγ); - Increases binding affinity to moderate affinity IL-2R receptor (IL-2Rβγ); - Reduced activation of CD25+ cells; - Reduces the stimulating effect of IL-2-mediated signaling in CD25+ and CD8+ T cells; - Removes or reduces the bias toward activation of CD25+ Treg cells; - It reduces the downregulation effect of the immune response by IL-2-induced Treg cells; -CD25- Maintains or enhances the activating effect of cells; and -CD25- increases the stimulation of IL-2-mediated proliferation and activation of effector T cells and NK cells; The mutant protein according to any one of claims 1 to 12.

14. It has one or more of the following characteristics: - In vivo antitumor effect; - No significant in vivo toxicity; and - Has storage stability; The mutant protein according to any one of claims 1 to 13.

15. A fusion protein of an IL-2 mutant protein, comprising the IL-2 mutant protein described in any one of claims 1 to 14.

16. The fusion protein according to claim 15, wherein the IL-2 mutant protein is fused with an Fc antibody fragment.

17. The fusion protein according to claim 16, wherein the IL-2 mutant protein is fused with Fc via a linker.

18. The fusion protein according to claim 17, wherein the linker is GSGS or 2x (G4S).

19. The fusion protein according to any one of claims 16 to 18, wherein the Fc is human IgG1Fc.

20. The fusion protein according to any one of claims 16 to 19, wherein the Fc comprises mutant L234A + L235A.

21. The aforementioned fusion protein (a) Sequence of sequence numbers 10, 11, 12, 13 or 14; (b) Sequence of sequence numbers 15, 16, 17, 18 or 19; or (c) A sequence having at least 95%, at least 96%, or 100% identity with the amino acid sequence of (a) or (b); A fusion protein according to any one of claims 16 to 19, comprising:

22. An immune complex comprising the IL-2 mutant protein and an antigen-binding molecule according to any one of claims 1 to 14.

23. The immunocomplex according to claim 22, wherein the antigen-binding molecule is an immunoglobulin molecule, or an antibody or antibody fragment.

24. IL-2 mutant protein according to any one of claims 1 to 14 an isolated polynucleotide encoding a fusion protein according to any one of claims 15 to 21, or an immune complex according to claim 22 or 23.

25. An expression vector comprising the polynucleotide described in claim 24.

26. A host cell comprising the polynucleotide described in claim 24 or the vector described in claim 25.

27. A method for producing an IL-2 mutant protein or its fusion protein or immune complex, comprising the step of culturing the host cells described in claim 26 under conditions suitable for the expression of the IL-2 mutant protein or its fusion protein or immune complex.

28. IL-2 mutant protein according to any one of claims 1 to 14 A pharmaceutical composition comprising a fusion protein according to any one of claims 15 to 21, or an immune complex according to claim 22 or 23, and a pharmaceutically acceptable carrier.

29. IL-2 mutant protein according to any one of claims 1 to 14 A pharmaceutical composition for use in the treatment of cancer, comprising a fusion protein according to any one of claims 15 to 21, or an immune complex according to claim 22 or 23.

30. A pharmaceutical composition for use in stimulating the immune system of a subject, comprising an IL-2 mutant protein according to any one of claims 1 to 11, a fusion protein according to any one of claims 12 to 19, or an immune complex according to claim 20 or 21.

31. A method for modifying the B'C' loop region of the wild-type IL-2 protein, which consists of amino acid residues from aa73 to aa83, (a) Substituting aa74 to aa83 in the B'C' loop region of the wild-type IL-2 protein with the sequence of GDASIH; or, (b) Substituting aa73 to aa83 in the B'C' loop region of the wild-type IL-2 protein with the sequence of AGDASIH; or, (c) The step of cleaving aa74 to aa83 from the C-terminus of the wild-type IL-2 protein B'C' loop region by 4 amino acids; or, (d) A step of replacing aa74 to aa83 in the B'C' loop region of the wild-type IL-2 protein with a sequence selected from the group consisting of QSKNFH, QSANFH, QSDNFH, GSKNFH, and QSANIH: Includes, Here, the positions of the amino acids are numbered according to Sequence ID No.

1. The aforementioned method.

32. - A step of introducing one or more mutations at the binding interface of IL-2 to IL-2Rα, - A step of expressing the modified IL-2 protein in the form of an Fc fusion in mammalian cells, - The following one or more improved characteristics: (i) Improved expression levels and / or protein purity, as measured by SEC-HPLC detection after one-step affinity chromatography; (ii) Reduced IL2Rα binding; (iii) Enhanced IL2Rβ binding; A step of identifying the modified protein having, The method according to claim 31, further comprising:

33. A modified IL-2 protein comprising a substitution in the sequence AGDASIH of the sequence from (aa)73 to (aa)83 of wild-type IL-2, compared to wild-type IL-2, wherein the amino acid positions are numbered according to SEQ ID NO: 1.

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