Interleukin-2 variant proteins promoting covalent chemical conjugation and uses thereof

Abstract

The present invention relates to mutant variants of IL-2 protein that promote chemical conjugation and their potential use as the treatment of cancer or inflammatory diseases.

Description

Technical Field

[0001] This disclosure relates to mutant variants of the IL-2 protein that promote chemical conjugation and their potential use as a treatment for cancer or inflammatory diseases. Background Technology

[0002] Achieving a viable therapeutic index in immunotherapy using systemically administered, natural, unmodified cytokines is notoriously difficult. This is due to the inherent high potency of cytokine molecules and the presence of a large number of responsive immune cells in disease-free tissues, some of which can produce different responses when bound by the same signaling molecules at any given concentration. Furthermore, inducing strong cytokine signaling in disease-free tissues often results in undesirable side effects.

[0003] Interleukin-2 (IL-2) is an example of a cytokine protein that regulates the activity of leukocytes (such as T cells and NK cells) responsible for immunity, responses to microbial infections, and the differentiation of foreign substances (“non-self”) from “self”. Therefore, IL-2 is a molecule with strong potential therapeutic value in both pro-inflammatory (e.g., in cancer) and anti-inflammatory (autoimmune diseases) environments. IL-2 modulates its action by binding to the IL-2 receptor complex, thereby stimulating the growth of immune cells (e.g., helper T cells, cytotoxic T cells, and regulatory T cells). The IL-2 receptor complex (IL-2R) consists of three chains, referred to as α (high affinity for CD25, no signal transduction), β (medium affinity for CD122, signal transduction), and γ (low affinity for CD132, signal transduction). Heterodimerization of the β and γ subunits of IL-2R is essential for signal transduction in T cells. Therefore, IL-2 can be mediated by the intermediate-affinity dimer CD122 / CD132IL-2R (Kd approximately 10). -9 M) or high-affinity trimers CD25 / CD122 / CD132 IL-2R (Kd approximately 10) -11 The IL-2R complex transmits signals. Different expression levels of these two forms of the IL-2R complex exist on NK cells, memory T cells, regulatory T cells, and activated T cells, leading to the pleiotrophic effect of IL-2, which depends on cytokine concentrations, the dominance of different cell populations, and the presence of alternative signals in any given tissue (disease- or normal). In cancer treatment with IL-2, overstimulation of regulatory T cells can suppress the desired stimulation of cytotoxic T effector cells. Similarly, rapid overstimulation of effector T cells throughout the system can lead to severe toxicity. The inherent complexity of IL-2 signaling across different cell classes increases the difficulty of using IL-2 as a soluble protein therapeutic agent.

[0004] To be used as potential therapeutic agents, IL-2 proteins are typically synthesized as free cytokines or antibody fusion proteins; however, these molecules are characterized by high toxicity, poor biodistribution, and / or poor therapeutic index. IL-2 and IL-2 antibody fusions can become more tolerable and potent therapeutic agents, for example, by improving their half-life in the human body while reducing their binding affinity to one or more of their three known receptors. This would allow for effective stimulation of desired effector cells of the immune system while minimizing stimulation of undesirable cells. For example, molecules with reduced or eliminated affinity for CD25 while having a conditional affinity for CD122 may exhibit a reduced ability to stimulate Treg cells (which are CD25-high) and more effectively stimulate Teff cells (CD25-low). This is a potentially beneficial phenotype in the cancer setting, where there is a desire to stimulate Teff cells more than Treg cells to stimulate anticancer therapeutic activity. In contrast, molecules with a natural (high) affinity for CD25 and a reduced affinity for CD122 exhibit an enhanced ability to stimulate Treg cells (which are CD25-high) and more effectively minimize stimulation of Teff cells (CD25-low). This is a potentially beneficial phenotype in inflammatory disease settings where stimulation of Treg cells over Teff cells is sought to stimulate anti-inflammatory therapeutic activity.

[0005] However, constructing IL-2-derived molecules with a suitable combination of expression characteristics, purification quality, stability, solubility, and controlled receptor signaling is no easy task. The ideal approach combines the ease of protein expression in a well-established CHO cell platform, purification via standard preparative column processes, and site-specific conjugation at optimal sites (or more) with a combination of regulation of receptor binding activity and pharmacokinetics. Therefore, there is a need for simplified and reproducible covalently modified forms of the IL-2 protein to produce molecules with particularly optimized activities to achieve the desired pharmacology in the diseased tissue setting.

[0006] The inventors have developed a novel mutant IL-2 derived from human IL-2, which contains a non-endogenous transglutaminase motif. This transglutaminase motif can be conjugated in a simple and reproducible manner via transglutaminase action. Summary of the Invention

[0007] This invention relates to mutant IL-2 derived from human IL-2 having the sequence SEQ ID NO: 1 or a variant thereof, wherein, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif is inserted by means of:

[0008] - Insert Q residues at one or more of the following positions in reference SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130, and / or

[0009] - Insert a motif at the N-terminus or C-terminus, wherein the motif comprises a sequence selected from the group consisting of or selected from the group consisting of LQS and TQG.

[0010] Optionally, with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0011] In one embodiment, the at least one non-endogenous transglutaminase motif is inserted by inserting a Q residue at one or more of the following positions in reference SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, S130.

[0012] In one embodiment, the at least one non-endogenous transglutaminase motif comprises or is composed of sequences selected from the group consisting of or selected from the following: X1LQ, where X1 is A, L, H, I, or V; NX2Q, where X2 is Y, H, or F; YRQ, LTQ; AQX3, where X3 is A or E; TEQ, LFQ; GSQ and VIQ, preferably selected from the group consisting of or selected from the following: X1LQ, where X1 is L, H, I, or V; NX2Q, where X2 is Y, H, or F; YRQ, LTQR (SEQ ID NO: 2); LFQ; GSQ and VIQ.

[0013] In one embodiment, the at least one non-endogenous transglutaminase motif comprises or is composed of the following:

[0014] - Selected from groups containing the following or sequences selected from the following: Preferably selected from the group consisting of the following or from the sequence of the following: And / or

[0015] - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the N-end, and / or

[0016] - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the C-terminus.

[0017] In one embodiment, the at least one non-endogenous transglutaminase motif is inserted by means of:

[0018] - Insert Q residues at one or more of the following positions in reference SEQ ID NO: 1: N33, T41, F42, H79, E100, and / or

[0019] - Insert a motif at the N-terminus or C-terminus, wherein the motif comprises or consists of a sequence selected from the group consisting of or selected from the group consisting of LQS and TQG.

[0020] In one embodiment, the at least one non-endogenous transglutaminase motif comprises or is composed of the following:

[0021] - Selected from groups containing the following or sequences selected from the following: And / or

[0022] - Selected from the group consisting of the following or the sequence consisting of the following: LQS and TQG, preferably wherein the sequence is added at the C-terminus or the N-terminus.

[0023] In one embodiment, the at least one non-endogenous transglutaminase motif comprises or is composed of the following:

[0024] - Selected from groups containing the following or sequences selected from the following: And / or

[0025] - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the N-end, and / or

[0026] - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the C-terminus.

[0027] In one embodiment, the mutant comprises or consists of sequences selected from the group consisting of or selected from the following: SEQ ID NO: 19 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63, SEQ ID NO: 110 to SEQ ID NO: 117, more preferably selected from the group consisting of or selected from the following: SEQ ID NO: 19 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63, and SEQ ID NO: 110 and 111.

[0028] In one embodiment, the mutant comprises or consists of sequences selected from the group consisting of or selected from the following: SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 29, SEQ ID NO: 31 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63, SEQ ID NO: 110 to SEQ ID NO: 117, more preferably selected from the group consisting of or selected from the following: SEQ ID NO: 19 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63, and SEQ ID NO: 110 and 111.

[0029] In one embodiment, the mutant comprises or consists of sequences selected from the group consisting of or selected from the following: SEQ ID NO: 26 to SEQ ID NO: 28, SEQ ID NO: 31 to SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 58 to SEQ ID NO: 63, and SEQ ID NO: 110 and 111.

[0030] In one embodiment, the mutant comprises or consists of sequences selected from the group consisting of or selected from the following: SEQ ID NO: 26 to SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO: 58 to SEQ ID NO: 63 and SEQ ID NO: 110 and 111.

[0031] In one embodiment, the mutant IL-2 further comprises replacing the F42 residue of reference SEQ ID NO: 1 with any other amino acid, more preferably replacing the F42 residue with an alanine residue.

[0032] The present invention also relates to a fusion protein comprising the mutant IL-2 as described above, and further comprising at least one masking portion, wherein the at least one masking portion is linked via a cleavable linker to at least one Q residue of a non-endogenous transglutaminase motif of the mutant IL-2.

[0033] In one embodiment, the masking portion is polyethylene glycol (PEG), preferably linear or branched PEG.

[0034] In one embodiment, the fusion protein further comprises an antibody or an antigen-binding fragment thereof or an antibody mimic, preferably wherein the antibody or an antigen-binding fragment thereof or an antibody mimic is linked to the mutant IL-2 via a linker.

[0035] This invention also relates to the fusion protein described above, which is used as a drug.

[0036] This invention also relates to the fusion protein described above, which is used to treat cancer or inflammatory diseases.

[0037] definition

[0038] In this invention, the following terms have the following meanings:

[0039] The "approximately / about" preceding a number covers a value plus or minus 10% or less. It should be understood that the value referred to by the term "approximately / about" is also specifically and preferably disclosed.

[0040] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to proteins having a combination of two heavy chains and two light chains, regardless of whether they possess any associated specific immunoreactivity. An "antibody" is a combination that has a significant, known specific immunoreactive activity against a target antigen. Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent bonds. The basic immunoglobulin structures in vertebrate systems are relatively well understood. The general term "immunoglobulin" encompasses five distinct classes of antibodies that can be distinguished biochemically. While the following discussion generally refers to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of this invention. Regarding IgG, immunoglobulins comprise two identical light polypeptide chains of approximately 23 kDa and two identical heavy chains of approximately 53 to 70 kDa. These four chains are linked by disulfide bonds in a "Y" configuration, where the light chain begins at the mouth of the "Y" and continues through a variable region to bracket the heavy chain. The light chains of antibodies are classified as kappa (κ) or lambda (λ). Each heavy chain class can be bonded to either a κ or λ light chain. Generally, the light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically modified host cells, the “tail” regions of the two heavy chains are bonded to each other via covalent disulfide or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the Y-configuration bifurcation end to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), with several subclasses (e.g., γ1 to γ4). The properties of this chain determine the “class” of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses or “isotypes” (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) have been well characterized and are known to confer functional specialization. In view of this disclosure, those skilled in the art can readily identify modified forms of each of these categories and isotypes, and therefore those within the scope of this invention. As described above, the variable region of the antibody allows the antibody to selectively recognize and specifically bind to epitopes on antigens. In other words, the variable domains of the light chain (VL domain) and the variable domains of the heavy chain (VH domain) of the antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site present at the end of each arm of the “Y”. More specifically, the antigen-binding site is defined by three complementarity determining regions (CDRs) on each VH and VL chain.

[0041] As used herein, "antigen-binding fragment" refers to a portion or region of an antibody that contains fewer amino acid residues than the complete antibody. An "antigen-binding fragment" binds to an antigen and / or competes with the complete antibody from which it is derived for antigen binding (e.g., specific binding to human CD25). Antigen-binding fragments encompass, but are not limited to, single-chain antibodies, dimer single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, nanobodies, domain antibodies, and unibody.

[0042] As used in this article, the term "derived from" indicates the relationship between the first and second molecules. It generally refers to the structural similarity between the first and second molecules and does not imply or include any restriction on the process or source of the first molecule derived from the second molecule. For example, in the case of mutant IL-2 derived from human IL-2, the mutant retains sufficient IL-2 structure to enable it to perform its necessary function—the ability to generate a signal under appropriate conditions. It does not imply or include any restriction on the specific process that produces mutant IL-2.

[0043] As used herein, "biantibody" refers to a small antibody fragment prepared by constructing an scFv fragment with a short linker (approximately 5 to 10 residues) between VH and VL, enabling interchain rather than intrachain pairing of variable domains, thereby producing a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific biantibodies are heterodimers of two "crossed" scFv fragments, where the VH and VL of the two antibodies are located on different polypeptide chains. For example, biantibodies are described in patent EP0404097 or patent application WO1993011161.

[0044] "Domain antibody" refers to the smallest functional binding unit of an antibody, corresponding to the variable region of the antibody heavy or light chain.

[0045] “Fab” refers to a monovalent fragment containing the following regions: VH, VL, CH1, and CL, which are linked by intramolecular disulfide bonds.

[0046] In this article, “F(ab')2” refers to a fragment containing two antigen-binding regions linked by disulfide bonds.

[0047] In this paper, "Fab" refers to the fragment obtained by restoring the F(ab')2 fragment.

[0048] "Fd fragment" refers to the heavy chain of the Fab fragment, which contains the VH and CH1 regions.

[0049] As used in this article, "Fv" refers to the smallest antibody fragment containing both a complete antigen recognition and binding site. This fragment consists of a VH and a VL dimer tightly non-covalently associated. The folding of these two domains produces six hypervariable rings (three rings each in the heavy and light chains), which facilitate antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three CDRs specific to the antigen) can recognize and bind antigens, although with lower affinity than the complete binding site.

[0050] As used herein, "humanized antibody or antigen-binding fragment thereof" refers to a chimeric antibody or antigen-binding fragment thereof comprising a minimal sequence derived from a non-human immunoglobulin. This includes antibodies produced by non-human cells having variable and constant regions that have been modified to more closely resemble antibodies produced by human cells, for example, by altering the amino acid sequence of the non-human antibody to incorporate amino acids present in human germline immunoglobulin sequences. Humanized antibodies or antigen-binding fragments thereof included in the fusion protein according to the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo), for example, in the CDR. The term "humanized antibody or antigen-binding fragment thereof" also includes antibodies and antigen-binding fragments thereof in which a CDR sequence derived from another mammalian species (e.g., mouse) has been branched onto a human frame sequence. In other words, the term "humanized antibody or antigen-binding fragment thereof" can refer to antibodies or antigen-binding fragments thereof in which the CDR of a recipient human antibody is replaced by a CDR from a donor non-human antibody. Humanized antibodies or their antigen-binding fragments may also include donor-derived residues in the frame sequence. Humanized antibodies or their antigen-binding fragments may also contain at least a portion of the constant region of human immunoglobulins. Humanized antibodies or their antigen-binding fragments may also contain residues that are not present in the recipient antibody or in the input CDR or frame sequence. Humanization can be performed using methods known in the art (e.g., Jones et al., 1986. Nature. 321(6069):522-5; Riechmann et al., 1988. Nature. 332(6162):323-7; Verhoeyen et al., 1988. Science. 239(4847):1534-6; Presta, 1992. Curr OpinBiotechnol. 3(4):394-8; US patent 4,816,567), methods including techniques such as “hyperhumanized” antibodies (e.g., Tan et al., 2002. J Immunol. 169(2):1119-25) and “surface remodeling” (e.g., Staelens et al., 2006. Mol Immunol. 43(8):1243-57; Roguska et al., 1994. Proc Natl Acad Sci USA. 91(3):969-73).

[0051] When used in this text to refer to the relationship between two or more amino acid sequences or two or more nucleic acid sequences, "identity" or "identical" refers to the degree of sequence correlation between the amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. In cases where gap alignment (if any) is handled by a specific mathematical model or computer program (i.e., an "algorithm"), "identity" measures the percentage of identical matches between the smaller sequences of the two or more sequences. The identity of related amino acid sequences or nucleic acid sequences can be readily calculated using known methods. Such methods include, but are not limited to, those described below: Lesk AM (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith DW (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin AM & Griffin HG (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecularbiology: treasure trove or trivial pursuit. San Diego, CA: Academic Press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo et al., 1988. SIAM J Appl Math. 48(5):1073-82. The preferred method for determining identity is designed to give the maximum match between the test sequences. Methods for determining identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two sequences include the GCG package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. NucleicAcids Res. 12(1 Pt 1):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J MolBiol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0052] Unless otherwise stated, “interleukin-2” or “IL-2” means any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers unprocessed IL-2 as well as any form of IL-2 produced through cellular processing. The term also covers naturally occurring variants of IL-2 (e.g., splice variants or allelic variants) and truncated forms of IL-2. In one embodiment, IL-2 is human IL-2 having the sequence SEQ ID NO: 1. In another embodiment, IL-2 is truncated human IL-2 having the sequence SEQ ID NO: 50.

[0053] "Interleukin-2 receptor" or "IL-2R" refers to the heterotrimeric receptor for IL-2. This receptor is composed of different combinations of the following chains: an α (alpha) chain (also known as IL-2Rα, CD25, or Tac antigen), a β (beta) chain (also known as IL-2Rβ or CD122), and a γ (gamma) chain (also known as IL-2Rγ, commonly the gamma chain or CD132). Depending on the combination of these chains, IL-2 receptors with low, intermediate, or high affinity for IL-2 can be generated. Low-affinity IL-2 receptors are composed of the IL-2Rα chain. Intermediate-affinity IL-2 receptors are composed of the IL-2Rβ and IL-2Rγ chains. High-affinity IL-2 receptors are composed of the IL-2Rα, IL-2Rβ, and IL-2Rγ chains.

[0054] "IL-2 mutant" or "mutant IL-2" refers to IL-2 that shows modifications in sequence and / or functional properties.

[0055] "Mammal" means any mammal, including humans, non-human primates, livestock and farm animals, as well as zoo, sporting or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0056] A peptide is a linear polymer of amino acids consisting of at least two and fewer than 50 amino acids linked together by peptide bonds.

[0057] "Polyethylene glycol" or "PEG" refers to a biocompatible, synthetic hydrophilic polyether compound with the formula shown above, where n ≥ 4. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The term "PEG" generally refers to oligomers and polymers with a molecular weight below 20,000 g / mol, while "PEO" refers to polymers with a molecular weight above 20,000 g / mol. Unless otherwise stated, the term "PEG" as used herein includes both "PEG" and "PEO" compounds.

[0058] As used herein, “single-chain antibody” refers to any antibody or fragment thereof that is a protein having a primary structure comprising or consisting of: a sequence of uninterrupted, continuous amino acid residues, including but not limited to (1) a single-chain Fv molecule (scFv); (2) a single-chain protein containing only a light chain variable domain or a fragment thereof containing three CDRs of the light chain variable domain and without an associated heavy chain portion; and (3) a single-chain protein containing only a heavy chain variable region or a fragment thereof containing three CDRs of the heavy chain variable region and without an associated light chain portion.

[0059] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a V that contains a chain of amino acids linked together to form a single amino acid chain. H and V L Antibody fragments containing antibody domains. Preferably, the scFv amino acid sequence also includes V. H With V L The peptide linkers between the domains enable scFv to form the desired structure for antigen binding.

[0060] "Small organic molecules" refers to molecules that are comparable in size to those organic molecules commonly used in pharmaceuticals. This term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules have a size range of up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0061] As used herein, “object” refers to a mammal, preferably a human. In one implementation, the object may be a “patient,” i.e., a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care, or who has been / is / will be the object of a medical procedure, or who is being monitored for the occurrence of a disease.

[0062] In the context of a constant region of a humanized or chimeric antibody or its antigen-binding fragment, "substantially human" means having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity with a human constant region. In this context, the term "human amino acid sequence" refers to the amino acid sequence encoded by human immunoglobulin genes, including germline, rearranged, and somatic mutation genes. The invention also contemplates proteins comprising a constant domain of a "human" sequence altered relative to a human sequence by the addition, deletion, or substitution of one or more amino acids, except for embodiments that explicitly require the presence of a "fully human hinge region." The presence of a "fully human hinge region" in at least one antibody or its antigen-binding fragment contained in a fusion protein according to the invention can be beneficial for both minimizing immunogenicity and optimizing antibody stability. It is considered that one or more amino acid substitutions, insertions, or deletions can be made within the constant region of the heavy chain and / or light chain, particularly within the Fc region. Amino acid substitutions can result in the substituted amino acid being replaced by a different naturally occurring amino acid, or by a non-natural or modified amino acid. It also allows for other structural modifications, such as changes in glycosylation patterns (e.g., by adding or deleting N- or O-linked glycosylation sites).

[0063] "Therapeutic effective dose" refers to the level or amount of antibody described herein, intended to (1) delay or prevent the onset of a disease, disorder, or condition, without causing significant negative or adverse side effects on the target; (2) slow or stop the progression, aggravation, or worsening of one or more symptoms of a disease, disorder, or condition; (3) improve the symptoms of a disease, disorder, or condition; (4) reduce the severity or incidence of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition. For prophylactic or preventative purposes, a therapeutic effective dose may be administered before the onset of a disease, disorder, or condition. Alternatively or as a supplement, for therapeutic purposes, a therapeutic effective dose may be administered after the onset of a disease, disorder, or condition.

[0064] "Treatment," "treatment," or "mitigation" refers to both therapeutic treatment and preventative or preventive measures; the aim of which is to prevent or slow down (mitigate) a target pathological symptom or disorder. Those requiring treatment include those already suffering from the disorder, those susceptible to the disorder, or those with a disorder to be prevented. In one embodiment, a subject's cancer is considered successfully "treated" if, after receiving a therapeutic dose of at least one of the mutant IL-2, fusion protein, nucleic acid, or expression vector according to the invention, the subject exhibits at least one of the following: a reduction in the number of cancer cells (or tumor size) or pathogenic cells; a reduction in the percentage of total cancerous or pathogenic cells; partial relief of one or more symptoms associated with the cancer or infectious disease to be treated; a reduction in morbidity and mortality; and an improvement in quality of life. The parameters used above to assess successful treatment and improvement of the disease can be easily measured using routine methods familiar to physicians.

[0065] "Unibody" refers to an antibody fragment that lacks the hinge region of an IgG4 antibody. The absence of the hinge region results in a molecule that is essentially half the size of a conventional IgG4 antibody and has a monovalent binding region instead of the bivalent binding region of an IgG4 antibody.

[0066] "Variation": refers to a nucleic acid or amino acid sequence that differs from the specific nucleic acid or amino acid sequence disclosed herein, typically having one or more substitutions, deletions, additions, and / or insertions. When referring to percentage of identity, it means a nucleic acid or amino acid sequence that contains at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the reference nucleic acid or amino acid sequence. Detailed Implementation Plan

[0067] This invention relates to mutants of IL-2 containing at least one transglutaminase site (TG), which enables conjugation of the conjugated site in a simple and reproducible manner via transglutaminase action. However, it should be understood that this concept of adding a TG site to the conjugated site can also be applied to other molecules, such as IL-2 mimics. Furthermore, it should be understood that other types of conjugation via non-natural amino acids and the introduction of other types of coupling sites can be applied to the sites identified herein.

[0068] The mutant IL-2 of this invention can exhibit reduced or eliminated affinity for CD25, while simultaneously exhibiting an affinity for the regulation of CD122, wherein the affinity for the regulation of CD122 is achieved through transglutaminase-mediated conjugation at one or more single sites, said sites being newly introduced into the IL-2 structure through mutation. The mutant IL-2 can be conjugated to a portion (e.g., PEG) via a selectively cleaved or non-selectively cleaved linker in diseased tissue. Without wishing to be bound by any theory, it is hypothesized that this form of mutant IL-2 can exhibit a reduced ability to stimulate regulatory T (Treg) cells (which are CD25-high) while effectively stimulating effector T (Teff) cells (CD25-low). This is a potentially beneficial phenotype in a cancer setting where there is a desire to stimulate Teff cells more than Treg cells, thereby stimulating anticancer therapeutic activity.

[0069] Alternatively, the mutant IL-2 of this invention may exhibit a natural (high) affinity for CD25, while simultaneously exhibiting a regulatory affinity for CD122, wherein the regulatory affinity for CD122 is achieved through transglutaminase-mediated conjugation at one or more single sites, said sites being novelly introduced into the IL-2 structure through mutation. The mutant IL-2 may conjugate with a portion (e.g., PEG) via a selectively cleaved or non-selectively cleaved linker in diseased tissue. Without wishing to be bound by any theory, it is hypothesized that the mutant IL-2 may exhibit an enhanced ability to stimulate Treg cells (which are CD25-high) and minimize stimulation of Teff cells (CD25-low). This is a potentially beneficial phenotype in inflammatory disease settings where stimulation of Treg cells over Teff cells is sought to stimulate anti-inflammatory therapeutic activity.

[0070] In one embodiment, the present invention relates to mutant IL-2, preferably mutant IL-2 derived from human IL-2.

[0071] In one embodiment, human IL-2 comprises or consists of the following: a sequence having SEQ ID NO: 1 or a variant thereof, preferably wherein the variant comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 1. Therefore, in one embodiment, the mutant IL-2 of the present invention is derived from human IL-2 having SEQ ID NO: 1 or a variant thereof, preferably wherein the variant comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 1.

[0072] The variant may contain the same number of amino acids as SEQ ID NO: 1, and therefore the mutations and positions described herein are identical for that variant. Alternatively, the variant may contain a different number of amino acids than SEQ ID NO: 1. In this case, those skilled in the art will know how to incorporate the mutations and positions described herein into the variant.

[0073] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation).

[0074] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamic acid residue (i.e., the Q74E mutation).

[0075] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0076] In one implementation, the IL-2 mutant exhibits reduced binding or affinity for the IL-2Rα chain compared to the derived IL-2.

[0077] In one implementation, the IL-2 mutant includes one or more of the following modifications:

[0078] - Referring to SEQ ID NO: 1, replace the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and / or

[0079] - Referring to SEQ ID NO: 1, replace the glutamine residue at position 74 with any other amino acid residue, preferably a glutamic acid residue (i.e., the Q74E mutation), and / or

[0080] - Referring to SEQ ID NO: 1, replace the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0081] In one embodiment, the mutant IL-2 contains at least one non-endogenous transglutaminase motif.

[0082] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises at least one non-endogenous transglutaminase motif and the phenylalanine residue at position 42 is replaced by any other amino acid residue, preferably by an alanine residue (i.e., the F42A mutation). Therefore, the invention also relates to a mutant IL-2, preferably derived from human IL-2 having SEQ ID NO: 1 or a variant thereof, referring to SEQ ID NO: 1, said mutant IL-2 comprising at least one non-endogenous transglutaminase motif and further comprising replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0083] In one embodiment, the mutant IL-2 contains a non-endogenous transglutaminase motif. In another embodiment, the mutant IL-2 contains two or more non-endogenous transglutaminase motifs.

[0084] As used herein, a transglutaminase motif is a motif containing at least one Q residue, particularly a surface-exposed Q residue, which is recognized by a transglutaminase (i.e., the side chain acts as a substrate for the transglutaminase), which catalyzes the formation of an isopeptide bond between the γ-carboxamide group (-(C=O)NH2) of the glutamine residue side chain and the ε-amino group (-NH2) of several primary amines (acyl acceptors), particularly the lysine residue side chain, and subsequently releases ammonia (NH3). Transglutaminase motifs can be endogenously present in proteins, and particularly in IL-2. For example, human IL-2 having SEQ ID NO: 1 contains an endogenous transglutaminase motif having a Q74 residue, and this motif consists of AQS.

[0085] As used in this article, the non-endogenous transglutaminase motif is the transglutaminase motif that is not present in IL-2 derived from IL-2 mutants.

[0086] In one embodiment, mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof does not contain a non-endogenous transglutaminase motif that is different from the non-endogenous transglutaminase motif described herein.

[0087] Therefore, in one embodiment, the mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof contains one or more non-endogenous transglutaminase motifs as described herein, and does not contain other non-endogenous transglutaminase motifs.

[0088] In one embodiment, the mutant IL-2 contains at least one non-endogenous transglutaminase motif inserted into the IL-2 sequence.

[0089] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting a glutamate (Q) residue at one or more of the following positions in reference SEQ ID NO:1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130.

[0090] Therefore, in one embodiment, at least one non-endogenous transglutaminase motif is characterized by the presence of glutamate (Q) residues at one or more of the following positions as referenced in SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130.

[0091] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting a glutamate (Q) residue at one or more of the following positions in reference SEQ ID NO:1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, and S130.

[0092] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting a glutamate (Q) residue at one or more of the following positions in reference SEQ ID NO:1: N33, T41, F42, H79, and E100.

[0093] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by replacing one or more of the following amino acids of reference SEQ ID NO: 1 with a Q residue: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, H79, E95, E100, E110, and S130.

[0094] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by replacing one or more of the following amino acids of reference SEQ ID NO: 1 with a Q residue: H16, L18, L19, N26, K32, N33, T41, F42, H79, E95, E100, and S130.

[0095] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by modifying the amino acid surrounding the Q57 residue of reference SEQ ID NO: 1, particularly one or more amino acids surrounding the Q57 residue. In this embodiment, the amino acid surrounding the Q57 residue is mutated, preferably replaced by other amino acids, to form the transglutaminase motif.

[0096] Therefore, in one embodiment, at least one non-endogenous transglutaminase motif is inserted via the following:

[0097] - Replace the Q residue at one or more of the following positions in reference SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, H79, E95, E100, and S130, and / or

[0098] - Replace one or more amino acids surrounding the Q57 residue of reference SEQ ID NO: 1 to form a transglutaminase motif.

[0099] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by replacing the amino acid H55 of reference SEQ ID NO: 1 with a leucine residue at the Q57 position (i.e., the H55L mutation).

[0100] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: X1LQ, where X1 is A, L, H, I or V; NX2Q, where X2 is Y, H or F; YRQ, LTQ; AQX3, where X3 is A or E, TEQ, LFQ; GSQ and VIQ.

[0101] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: X1LQ, where X1 is L, H, I or V; NX2Q, where X2 is Y, H or F; YRQ, LTQR (SEQ ID NO: 2); LFQ; GSQ and VIQ.

[0102] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the group consisting of LLQ, YRQ, LTQR (SEQ ID NO: 2), LFQ, and GSQ.

[0103] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: .

[0104] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: .

[0105] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: .

[0106] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting glutamate (Q) residues at one or more of the following positions as referenced in SEQ ID NO:1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130, and comprises or consists of sequences selected from the group consisting of or selected from the following: X1LQ, where X1 is A, L, H, I, or V; NX2Q, where X2 is Y, H, or F; YRQ, LTQ; AQX3, where X3 is A or E; TEQ; LFQ; GSQ, and VIQ.

[0107] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting glutamate (Q) residues at one or more of the following positions in reference SEQ ID NO:1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130, and comprises or consists of sequences selected from the group consisting of or selected from the following: .

[0108] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting glutamate (Q) residues at one or more of the following positions as referenced in SEQ ID NO:1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, and S130, and comprises or consists of sequences selected from the group consisting of or selected from the following: X1LQ, where X1 is L, H, I, or V; NX2Q, where X2 is Y, H, or F; YRQ; LTQR (SEQ ID NO:2); LFQ; GSQ, and VIQ.

[0109] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting glutamate (Q) residues at one or more of the following positions in reference SEQ ID NO:1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, and S130, and comprises or consists of sequences selected from the group consisting of or selected from the following: .

[0110] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting glutamate (Q) residues at one or more of the following positions in reference SEQ ID NO:1: N33, T41, F42, H79, and E100, and comprises or consists of sequences selected from the group consisting of or selected from the group consisting of: LLQ, YRQ, LTQR (SEQ ID NO: 2), LFQ, and GSQ.

[0111] In one embodiment, at least one non-endogenous transglutaminase motif is inserted by inserting glutamate (Q) residues at one or more of the following positions in reference SEQ ID NO:1: N33, T41, F42, H79, and E100, and comprises or consists of a sequence selected from the group consisting of or selected from the following: .

[0112] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0113] - Inserted by inserting a Q residue (i.e., containing a Q19 residue, preferably obtained via an L19Q mutation) at the L19 position of reference SEQ ID NO: 1, and

[0114] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LEHLLQD (SEQ ID NO: 3),

[0115] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0116] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 19. An example of such a mutant IL-2 is mutant 2-202.

[0117]

[0118] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 155. An example of such a mutant IL-2 is mutant 2-596.

[0119]

[0120] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0121] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 130. An example of such a mutant IL-2 is mutant 2-572.

[0122]

[0123] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0124] - Inserted by inserting a Q residue (i.e., containing a Q18 residue, preferably obtained via an L18Q mutation) at the L18 position of reference SEQ ID NO: 1, and

[0125] - Contains or consists of the sequence HLQ, preferably contains or consists of the sequence LEHLQSD (SEQ ID NO: 4),

[0126] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0127] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 20. An example of such a mutant IL-2 is mutant 2-203.

[0128]

[0129] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 156. An example of such a mutant IL-2 is mutant 2-597.

[0130]

[0131] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0132] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 131. An example of such a mutant IL-2 is mutant 2-573.

[0133]

[0134] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0135] - Inserted by inserting a Q residue (i.e., containing a Q16 residue, preferably obtained through an H16Q mutation) at the H16 position of reference SEQ ID NO: 1, and

[0136] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQLLLD (SEQ ID NO: 5),

[0137] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0138] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 21. An example of such a mutant IL-2 is mutant 2-204.

[0139]

[0140] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 157. An example of such a mutant IL-2 is mutant 2-598.

[0141]

[0142] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0143] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 132. An example of such a mutant IL-2 is mutant 2-574.

[0144]

[0145] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0146] - Inserted by inserting a Q residue (i.e., containing a Q26 residue, preferably obtained via an N26Q mutation) at the N26 position of reference SEQ ID NO: 1, and

[0147] - Contains or consists of the sequence ILQ, preferably contains or consists of MILQGIN (SEQ ID NO: 6),

[0148] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0149] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 22. An example of such a mutant IL-2 is mutant 2-205.

[0150]

[0151] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 158. An example of such a mutant IL-2 is mutant 2-599.

[0152]

[0153] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0154] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 133. An example of such a mutant IL-2 is mutant 2-575.

[0155]

[0156] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0157] - Inserted by inserting a Q residue (i.e., containing a Q26 residue, preferably obtained via an N26Q mutation) at the N26 position of reference SEQ ID NO: 1, and

[0158] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence MLLQGIN (SEQ ID NO: 7),

[0159] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0160] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 23. An example of such a mutant IL-2 is mutant 2-206.

[0161]

[0162] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 159. An example of such a mutant IL-2 is mutant 2-600.

[0163]

[0164] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0165] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 134. An example of such a mutant IL-2 is mutant 2-576.

[0166]

[0167] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0168] - Inserted by inserting a Q residue (i.e., containing a Q32 residue, preferably obtained via a K32Q mutation) at the K32 position of reference SEQ ID NO: 1, and

[0169] - Contains or consists of the sequence NYQ, preferably contains or consists of the sequence NNYQNP (SEQ ID NO: 8),

[0170] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0171] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 24. An example of such a mutant IL-2 is mutant 2-207.

[0172]

[0173] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 160. An example of such a mutant IL-2 is mutant 2-601.

[0174]

[0175] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0176] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 135. An example of such a mutant IL-2 is mutant 2-577.

[0177]

[0178] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0179] - Inserted by inserting a Q residue (i.e., containing a Q32 residue, preferably obtained via a K32Q mutation) at the K32 position of reference SEQ ID NO: 1, and

[0180] - Contains or consists of the sequence NHQ, preferably contains or consists of the sequence NNHQNP (SEQ ID NO: 9),

[0181] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0182] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 25. An example of such a mutant IL-2 is mutant 2-208.

[0183]

[0184] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 161. An example of such a mutant IL-2 is mutant 2-602.

[0185]

[0186] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0187] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 136. An example of such a mutant IL-2 is mutant 2-578.

[0188]

[0189] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0190] - Inserted by inserting a Q residue (i.e., containing a Q33 residue, preferably obtained via an N33Q mutation) at the N33 position of reference SEQ ID NO: 1, and

[0191] - Contains or consists of the sequence YRQ, preferably contains or consists of the sequence NNYRQP (SEQ ID NO: 102),

[0192] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0193] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 110. An example of such a mutant IL-2 is mutant 2-103.

[0194]

[0195] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 162. An example of such a mutant IL-2 is mutant 2-603.

[0196]

[0197] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0198] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 137. An example of such a mutant IL-2 is mutant 2-579.

[0199]

[0200] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0201] - Inserted by inserting a Q residue (i.e., containing a Q33 residue, preferably obtained via an N33Q mutation) at the N33 position of reference SEQ ID NO: 1, and

[0202] - Contains or consists of the sequence YRQ, preferably contains or consists of the sequence NRYRQP (SEQ ID NO: 103),

[0203] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0204] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 111. An example of such a mutant IL-2 is mutant 2-104.

[0205]

[0206] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 163. An example of such a mutant IL-2 is mutant 2-604.

[0207]

[0208] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0209] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 138. An example of such a mutant IL-2 is mutant 2-580.

[0210]

[0211] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0212] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0213] - Contains or consists of the sequence LLQ, preferably contains or consists of the preferred sequence LLQFKF (SEQ ID NO: 10),

[0214] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0215] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 26. An example of such a mutant IL-2 is mutant 2-105.

[0216]

[0217] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 164. An example of such a mutant IL-2 is mutant 2-605.

[0218]

[0219] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0220] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 139. An example of such a mutant IL-2 is mutant 2-581.

[0221]

[0222] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0223] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0224] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11),

[0225] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0226] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 27. An example of such a mutant IL-2 is mutant 2-106.

[0227]

[0228] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 62. An example of such a mutant IL-2 is mutant 2-130.

[0229]

[0230] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0231] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0232] - Contains or consists of the following: sequence LTQ, particularly LTQR (SEQ ID NO: 2), preferably contains or consists of sequence MLTQRF (SEQ ID NO: 12),

[0233] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0234] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 28. An example of such a mutant IL-2 is mutant 2-107.

[0235]

[0236] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 63. An example of such a mutant IL-2 is mutant 2-131.

[0237]

[0238] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0239] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0240] - Contains or consists of the following: the sequence LTQ, particularly LTQG (SEQ ID NO: 118), preferably containing or consisting of the sequence MLTQGF (SEQ ID NO: 104),

[0241] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0242] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 112. An example of such a mutant IL-2 is mutant 2-108.

[0243]

[0244] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 165. An example of such a mutant IL-2 is mutant 2-606.

[0245]

[0246] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0247] - Inserted by inserting a Q residue (i.e., containing a Q51 residue, preferably obtained via a T51Q mutation) at the T51 position of reference SEQ ID NO: 1, and

[0248] - Contains or consists of the sequence AQE, preferably contains or consists of the sequence LAQELK (SEQ ID NO: 105),

[0249] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0250] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 113. An example of such a mutant IL-2 is mutant 2-109.

[0251]

[0252] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 166. An example of such a mutant IL-2 is mutant 2-607.

[0253]

[0254] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0255] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 140. An example of such a mutant IL-2 is mutant 2-582.

[0256]

[0257] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0258] - Inserted by inserting a Q residue (i.e., containing a Q51 residue, preferably obtained via a T51Q mutation) at the T51 position of reference SEQ ID NO: 1, and

[0259] - Contains or consists of the sequence AQA, preferably contains or consists of the sequence LAQALK (SEQ ID NO: 106).

[0260] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0261] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 114. An example of such a mutant IL-2 is mutant 2-110.

[0262]

[0263] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 167. An example of such a mutant IL-2 is mutant 2-608.

[0264]

[0265] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0266] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 141. An example of such a mutant IL-2 is mutant 2-583.

[0267]

[0268] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0269] - Inserted by inserting a Q residue (i.e., containing a Q53 residue, preferably obtained via an L53Q mutation) at the L53 position of reference SEQ ID NO: 1, and

[0270] - Contains or consists of the sequence TEQ, preferably contains or consists of the sequence KATEQK (SEQ ID NO: 107),

[0271] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0272] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 115. An example of such a mutant IL-2 is mutant 2-111.

[0273]

[0274] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 168. An example of such a mutant IL-2 is mutant 2-609.

[0275]

[0276] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0277] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 142. An example of such a mutant IL-2 is mutant 2-584.

[0278]

[0279] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0280] - Inserted by inserting a Q residue (i.e., containing a Q57 residue) at the Q57 position of reference SEQ ID NO: 1, and

[0281] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQC (SEQ ID NO: 13),

[0282] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0283] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 29. An example of such a mutant IL-2 is mutant 2-209.

[0284]

[0285] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 169. An example of such a mutant IL-2 is mutant 2-610.

[0286]

[0287] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0288] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 143. An example of such a mutant IL-2 is mutant 2-585.

[0289]

[0290] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0291] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0292] - Contains or consists of the sequence NFQ, preferably contains or consists of the sequence KNFQLRPRD (SEQ ID NO: 14),

[0293] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0294] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 30. An example of such a mutant IL-2 is mutant 2-210.

[0295]

[0296] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 170. An example of such a mutant IL-2 is mutant 2-611.

[0297]

[0298] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0299] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 144. An example of such a mutant IL-2 is mutant 2-222.

[0300]

[0301] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0302] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0303] - Contains or consists of the sequence LFQ, preferably contains or consists of the sequence KLFQLRPRD (SEQ ID NO: 15),

[0304] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0305] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 31. An example of such a mutant IL-2 is mutant 2-112.

[0306]

[0307] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 32. An example of such a mutant IL-2 is mutant 2-114.

[0308]

[0309] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0310] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0311] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0312] - Contains or consists of the sequence LFQ, preferably contains or consists of the sequence KLFQLRPRD (SEQ ID NO: 15), and

[0313] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0314] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0315] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 64. An example of such a mutant IL-2 is mutant 2-132.

[0316]

[0317] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0318] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0319] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16),

[0320] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0321] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 33. An example of such a mutant IL-2 is mutant 2-113.

[0322]

[0323] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 34. An example of such a mutant IL-2 is mutant 2-115.

[0324]

[0325] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0326] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0327] - Inserted by inserting a Q residue at the H79 position of reference SEQ ID NO: 1 (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation), and

[0328] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16), and

[0329] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0330] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0331] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 65. An example of such a mutant IL-2 is mutant 2-133.

[0332]

[0333] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0334] - Inserted by inserting a Q residue (i.e., containing a Q95 residue, preferably obtained through an E95Q mutation) at the E95 position of reference SEQ ID NO: 1, and

[0335] - Contains or consists of the sequence VLQ.

[0336] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0337] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 35. An example of such a mutant IL-2 is mutant 2-211.

[0338]

[0339] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 171. An example of such a mutant IL-2 is mutant 2-612.

[0340]

[0341] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0342] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 145. An example of such a mutant IL-2 is mutant 2-586.

[0343]

[0344] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0345] - Inserted by inserting a Q residue (i.e., containing a Q95 residue, preferably obtained through an E95Q mutation) at the E95 position of reference SEQ ID NO: 1, and

[0346] - Contains or consists of the sequence LLQ.

[0347] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0348] In one embodiment, a mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof comprises or consists of a sequence having SEQ ID NO: 36. An example of such a mutant IL-2 is mutant 2-212.

[0349]

[0350] In one embodiment, a mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof comprises or consists of a sequence having SEQ ID NO: 172. An example of such a mutant IL-2 is mutant 2-613.

[0351]

[0352] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0353] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 146. An example of such a mutant IL-2 is mutant 2-587.

[0354]

[0355] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0356] - Inserted by inserting a Q residue (i.e., containing a Q100 residue, preferably obtained via an E100Q mutation) at the E100 position of reference SEQ ID NO: 1, and

[0357] - Contains or consists of the sequence GSQ, preferably contains or consists of the sequence GSQTT (SEQ ID NO: 17),

[0358] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0359] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 37. An example of such a mutant IL-2 is mutant 2-213.

[0360]

[0361] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 173. An example of such a mutant IL-2 is mutant 2-614.

[0362]

[0363] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0364] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 147. An example of such a mutant IL-2 is mutant 2-588.

[0365]

[0366] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0367] - Inserted by inserting a Q residue (i.e., containing a Q100 residue, preferably obtained via an E100Q mutation) at the E100 position of reference SEQ ID NO: 1, and

[0368] - Contains or consists of the sequence GSQ, preferably contains or consists of the sequence GSQST (SEQ ID NO: 18),

[0369] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0370] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 38. An example of such a mutant IL-2 is mutant 2-116.

[0371]

[0372] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 174. An example of such a mutant IL-2 is mutant 2-615.

[0373]

[0374] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0375] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0376] - Inserted by inserting a Q residue at the E100 position of reference SEQ ID NO: 1 (i.e., containing a Q100 residue, preferably obtained via an E100Q mutation), and

[0377] - Contains or consists of the sequence GSQ, preferably contains or consists of the sequence GSQST (SEQ ID NO: 18), and

[0378] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0379] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0380] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 66. An example of such a mutant IL-2 is mutant 2-134.

[0381]

[0382] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0383] - Insertion is achieved by inserting a Q residue (i.e., containing a Q110 residue, preferably obtained via an E110Q mutation) at the E110 position of reference SEQ ID NO: 1, and

[0384] - Contains or consists of the sequence ALQ, preferably contains or consists of the sequence EYALQ (SEQ ID NO: 108).

[0385] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0386] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 116. An example of such a mutant IL-2 is mutant 2-117.

[0387]

[0388] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 175. An example of such a mutant IL-2 is mutant 2-616.

[0389]

[0390] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0391] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 148. An example of such a mutant IL-2 is mutant 2-589.

[0392]

[0393] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0394] - Insertion is achieved by inserting a Q residue (i.e., containing a Q110 residue, preferably obtained via an E110Q mutation) at the E110 position of reference SEQ ID NO: 1, and

[0395] - Contains or consists of the sequence ALQ, preferably contains or consists of the sequence DYALQ (SEQ ID NO: 109),

[0396] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0397] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 117. An example of such a mutant IL-2 is mutant 2-118.

[0398]

[0399] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 176. An example of such a mutant IL-2 is mutant 2-617.

[0400]

[0401] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0402] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 149. An example of such a mutant IL-2 is mutant 2-590.

[0403]

[0404] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0405] - Inserted by inserting a Q residue (i.e., containing a Q130 residue, preferably obtained through an S130Q mutation) at the S130 position of reference SEQ ID NO: 1, and

[0406] - Contains or consists of the sequence VIQ,

[0407] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0408] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 39. An example of such a mutant IL-2 is mutant 2-214.

[0409]

[0410] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 177. An example of such a mutant IL-2 is mutant 2-618.

[0411]

[0412] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0413] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 150. An example of such a mutant IL-2 is mutant 2-591.

[0414]

[0415] In one embodiment, the mutant IL-2 of the present invention comprises at least one non-endogenous transglutaminase motif inserted at the N-terminus or C-terminus of the IL-2 sequence, wherein the mutant IL-2 is preferably derived from human IL-2 having SEQ ID NO: 1 or a variant thereof.

[0416] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the group consisting of LQS and TQG, preferably wherein the sequence is present at or added to the N-terminus or C-terminus.

[0417] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: Preferably, the sequence is present or added at the N-terminus or C-terminus.

[0418] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: Preferably, the sequence is present or added at the N-terminus.

[0419] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the following: Preferably, the sequence is present or added at the N-terminus.

[0420] In one embodiment, at least one non-endogenous transglutaminase motif defined above is inserted at the N-terminus of the IL-2 sequence having SEQ ID NO: 50.

[0421] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the group consisting of GGTQGA (SEQ ID NO: 46), GGLQSP (SEQ ID NO: 47), GLQSP (SEQ ID NO: 48), LQSP (SEQ ID NO: 49), and QGA, preferably wherein the sequence is present or is added at the C-terminus.

[0422] In one embodiment, at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group consisting of or selected from the group consisting of GGLQSP (SEQ ID NO: 47), GLQSP (SEQ ID NO: 48), LQSP (SEQ ID NO: 49), and QGA, preferably wherein the sequence is present or is added at the C-terminus.

[0423] In one embodiment, at least one non-endogenous transglutaminase motif defined above is inserted at the C-terminus of the IL-2 sequence having SEQ ID NO: 1.

[0424] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0425] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or a sequence consisting of LQS (i.e., containing a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or a sequence consisting of LQS).

[0426] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0427] In one embodiment, a sequence comprising LQS or thereof, preferably comprising LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 1.

[0428] In one embodiment, a sequence comprising LQS or thereof, preferably comprising LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0429] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 51. An example of such a mutant IL-2 is mutant 2-120.

[0430]

[0431] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 178. An example of such a mutant IL-2 is mutant 2-619.

[0432]

[0433] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0434] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0435] - Inserted by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or a sequence consisting of LQS (i.e., containing a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or a sequence consisting of LQS), and

[0436] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0437] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0438] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 67. An example of such a mutant IL-2 is mutant 2-135.

[0439]

[0440] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0441] - Insertion is achieved by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGAPTSSSTKKTQ (SEQ ID NO: 41) or consisting of TQG (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGAPTSSSTKKTQ (SEQ ID NO: 41) or consisting of TQG).

[0442] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0443] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGAPTSSSTKKTQ (SEQ ID NO: 41) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 1.

[0444] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGAPTSSSTKKTQ (SEQ ID NO: 41) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0445] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 52. An example of such a mutant IL-2 is mutant 2-215.

[0446]

[0447] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 179. An example of such a mutant IL-2 is mutant 2-620.

[0448]

[0449] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0450] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 151. An example of such a mutant IL-2 is mutant 2-592.

[0451]

[0452] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0453] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQS at the N-terminus (i.e., containing a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQS at the N-terminus).

[0454] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0455] In one embodiment, a sequence comprising LQS or thereof, preferably comprising LQSPTSSSTKKTQ (SEQ ID NO: 42) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 1.

[0456] In one embodiment, a sequence comprising LQS or thereof, preferably comprising LQSPTSSSTKKTQ (SEQ ID NO: 42) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0457] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 53. An example of such a mutant IL-2 is mutant 2-121.

[0458]

[0459] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 180. An example of such a mutant IL-2 is mutant 2-621.

[0460]

[0461] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0462] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0463] - Inserted by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or a sequence consisting of LQS (i.e., containing a sequence containing or consisting of LQS at the N-terminus, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or a sequence consisting of LQS), and

[0464] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0465] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0466] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 68. An example of such a mutant IL-2 is mutant 2-136.

[0467]

[0468] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0469] - Insertion is achieved by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGASSSTKKTQ (SEQ ID NO: 43) or consisting of TQG (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGASSSTKKTQ (SEQ ID NO: 43) or consisting of TQG).

[0470] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0471] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGASSSTKKTQ (SEQ ID NO: 43) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 1.

[0472] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGASSSTKKTQ (SEQ ID NO: 43) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0473] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 54. An example of such a mutant IL-2 is mutant 2-216.

[0474]

[0475] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 181. An example of such a mutant IL-2 is mutant 2-622.

[0476]

[0477] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0478] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 152. An example of such a mutant IL-2 is mutant 2-593.

[0479]

[0480] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0481] - Insertion is achieved by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or consisting of TQG (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or consisting of TQG).

[0482] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0483] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGASSTKKTQ (SEQ ID NO: 44) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 1.

[0484] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGASSTKKTQ (SEQ ID NO: 44) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0485] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 55. An example of such a mutant IL-2 is mutant 2-122.

[0486]

[0487] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 182. An example of such a mutant IL-2 is mutant 2-623.

[0488]

[0489] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0490] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0491] - Inserted by inserting a sequence containing TQG or composed thereof at the N-terminus, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or composed thereof (i.e., containing a sequence containing TQG or composed thereof at the N-terminus, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or composed thereof), and

[0492] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0493] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0494] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 69. An example of such a mutant IL-2 is mutant 2-137.

[0495]

[0496] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0497] - Insertion is achieved by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGASTKKTQ (SEQ ID NO: 45) or consisting of TQG (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing TQGASTKKTQ (SEQ ID NO: 45) or consisting of TQG).

[0498] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0499] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGASTKKTQ (SEQ ID NO: 45) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 1.

[0500] In one embodiment, a sequence comprising TQG or thereof, preferably comprising TQGASTKKTQ (SEQ ID NO: 45) or thereof, is inserted at the N-terminus of the sequence having SEQ ID NO: 50. An example of such a mutant IL-2 is mutant 2-217.

[0501] In one embodiment, the mutant IL-2 comprises or consists of the sequence having SEQ ID NO: 56. An example of such a mutant IL-2 is mutant 2-217.

[0502]

[0503] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 183. An example of such a mutant IL-2 is mutant 2-624.

[0504]

[0505] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0506] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 153. An example of such a mutant IL-2 is mutant 2-594.

[0507]

[0508] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0509] - Insertion is achieved by inserting a sequence containing TQG or composed thereof at the C-terminus, preferably containing GGTQGA (SEQ ID NO: 46) or composed thereof (i.e., the C-terminus contains a sequence containing TQG or composed thereof, preferably containing GGTQGA (SEQ ID NO: 46) or composed thereof).

[0510] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); and / or replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0511] In one embodiment, a sequence comprising TQG or thereof, preferably comprising GGTQGA (SEQ ID NO:46) or thereof, is inserted at the C-terminus of the sequence having SEQ ID NO:1.

[0512] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 57. An example of such a mutant IL-2 is mutant 2-218.

[0513]

[0514] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 184. An example of such a mutant IL-2 is mutant 2-625.

[0515]

[0516] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0517] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 154. An example of such a mutant IL-2 is mutant 2-595.

[0518]

[0519] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0520] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47) (i.e., containing a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47)).

[0521] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0522] In one embodiment, a sequence comprising LQS or thereof, preferably comprising GGLQSP (SEQ ID NO:47) or thereof, is inserted at the C-terminus of the sequence having SEQ ID NO:1.

[0523] In one embodiment, a sequence comprising LQS or thereof, preferably comprising GGLQSP (SEQ ID NO:47) or thereof, is inserted at the C-terminus of the sequence having SEQ ID NO:50.

[0524] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 58. An example of such a mutant IL-2 is mutant 2-123.

[0525]

[0526] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 185. An example of such a mutant IL-2 is mutant 2-626.

[0527]

[0528] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0529] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0530] - Inserted by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47) (i.e., containing a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47)), and

[0531] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0532] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0533] In one embodiment, the mutant IL- comprises or consists of a sequence having SEQ ID NO: 70. An example of such a mutant IL-2 is mutant 2-138.

[0534] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0535] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GLQSP (SEQ ID NO: 48) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GLQSP (SEQ ID NO: 48)).

[0536] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0537] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing GLQSP (SEQ ID NO: 48) or composed of GLQSP (SEQ ID NO: 48), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0538] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing GLQSP (SEQ ID NO: 48) or composed of GLQSP (SEQ ID NO: 48), is inserted at the C-terminus of the sequence having SEQ ID NO: 50.

[0539] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 59. An example of such a mutant IL-2 is mutant 2-124.

[0540] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 186. An example of such a mutant IL-2 is mutant 2-627.

[0541] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0542] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0543] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GLQSP (SEQ ID NO: 48) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GLQSP (SEQ ID NO: 48)).

[0544] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0545] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0546] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 122. An example of such a mutant IL-2 is mutant 2-139.

[0547] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0548] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of LQSP (SEQ ID NO: 49) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSP (SEQ ID NO: 49)).

[0549] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0550] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSP (SEQ ID NO: 49) or composed of LQSP (SEQ ID NO: 49), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0551] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSP (SEQ ID NO: 49) or composed of LQSP (SEQ ID NO: 49), is inserted at the C-terminus of the sequence having SEQ ID NO: 50.

[0552] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 60. An example of such a mutant IL-2 is mutant 2-125.

[0553] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 187. An example of such a mutant IL-2 is mutant 2-628.

[0554] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0555] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0556] - Inserted by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of LQSP (SEQ ID NO: 49) (i.e., containing a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of LQSP (SEQ ID NO: 49)), and

[0557] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0558] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0559] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 71. An example of such a mutant IL-2 is mutant 2-140.

[0560] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0561] - Insertion is achieved by inserting a sequence containing or consisting of QGA at the C end (i.e., the C end contains a sequence containing or consisting of QGA).

[0562] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0563] In one embodiment, a sequence containing or composed of QGAs is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0564] In one embodiment, a sequence containing or composed of QGAs is inserted at the C-terminus of the sequence having SEQ ID NO: 50.

[0565] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 61. An example of such a mutant IL-2 is mutant 2-126.

[0566] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 188. An example of such a mutant IL-2 is mutant 2-629.

[0567] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 comprises replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0568] Therefore, in one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein the motif:

[0569] - Insertion is achieved by inserting a sequence containing or consisting of QGAs at the C-end (i.e., the C-end contains a sequence containing or consisting of QGAs), and

[0570] Referring to SEQ ID NO: 1, the mutant IL-2 contains the substitution of the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0571] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0572] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 72. An example of such a mutant IL-2 is mutant 2-141.

[0573] The present invention also relates to a mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof, said mutant IL-2 containing at least two non-endogenous transglutaminase motifs.

[0574] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof further comprises replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation).

[0575] In one embodiment, referring to SEQ ID NO: 1, the mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof further comprises replacing the 74th glutamine residue with any other amino acid residue, preferably a glutamic acid residue (i.e., the Q74E mutation).

[0576] In one implementation, at least two non-endogenous transglutaminase motifs are defined as described above.

[0577] In one embodiment, one of the non-endogenous transglutaminase motifs includes the insertion of a Q residue at position T41 or F42 of reference SEQ ID NO: 1.

[0578] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0579] One of the motifs:

[0580] - Inserted by inserting a Q residue at the T41 position of reference SEQ ID NO: 1 (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation), and

[0581] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0582] Another motif:

[0583] - Inserted by inserting a Q residue at the H79 position of reference SEQ ID NO: 1 (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation), and

[0584] - Contains or consists of the sequence LFQ, preferably contains or consists of the sequence KLFQLRPRD (SEQ ID NO: 15),

[0585] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0586] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 73. An example of such a mutant IL-2 is mutant 2-142.

[0587] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0588] One of the motifs:

[0589] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0590] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0591] Another motif:

[0592] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0593] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16),

[0594] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0595] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 74. An example of such a mutant IL-2 is mutant 2-143.

[0596] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0597] One of the motifs:

[0598] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0599] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0600] Another motif:

[0601] - Inserted by inserting a Q residue (i.e., containing a Q100 residue, preferably obtained via an E100Q mutation) at the E100 position of reference SEQ ID NO: 1, and

[0602] - Contains or consists of the sequence GSQ, preferably contains or consists of the sequence GSQST (SEQ ID NO: 18),

[0603] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0604] In one embodiment, a mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof comprises or consists of a sequence having SEQ ID NO: 75. An example of such a mutant IL-2 is mutant 2-144.

[0605] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0606] One of the motifs:

[0607] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0608] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0609] Another motif:

[0610] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or consisting of the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) (i.e., the N-terminus contains a sequence containing or consisting of LQS, preferably containing LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or consisting of the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40)).

[0611] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0612] In one embodiment, a sequence containing or composed of LQS, preferably containing or composed of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or composed of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0613] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 76. An example of such a mutant IL-2 is mutant 2-145.

[0614] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0615] One of the motifs:

[0616] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0617] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0618] Another motif:

[0619] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) (i.e., the N-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42)).

[0620] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0621] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or composed of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0622] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 77. An example of such a mutant IL-2 is mutant 2-146.

[0623] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0624] One of the motifs:

[0625] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0626] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0627] Another motif:

[0628] - Inserted by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing or consisting of TQG (SEQ ID NO: 44) (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing or consisting of TQG (SEQ ID NO: 44)).

[0629] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0630] In one embodiment, a sequence containing TQG or consisting of TQG, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or consisting of TQGASSTKKTQ (SEQ ID NO: 44), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0631] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 78. An example of such a mutant IL-2 is mutant 2-147.

[0632] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0633] One of the motifs:

[0634] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0635] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0636] Another motif:

[0637] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of LQSP (SEQ ID NO: 49) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSP (SEQ ID NO: 49)).

[0638] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0639] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSP (SEQ ID NO: 49) or composed of LQSP (SEQ ID NO: 49), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0640] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 79. An example of such a mutant IL-2 is mutant 2-150.

[0641] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0642] One of the motifs:

[0643] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0644] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0645] Another motif:

[0646] - Insertion is achieved by inserting a sequence containing or consisting of QGA at the C end (i.e., the C end contains a sequence containing or consisting of QGA).

[0647] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0648] In one embodiment, a sequence containing or composed of QGAs is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0649] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 80. An example of such a mutant IL-2 is mutant 2-151.

[0650] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0651] One of the motifs:

[0652] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0653] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0654] Another motif:

[0655] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0656] - Contains or consists of the sequence LFQ, preferably contains or consists of the sequence KLFQLRPRD (SEQ ID NO: 15),

[0657] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0658] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 81. An example of such a mutant IL-2 is mutant 2-152.

[0659] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0660] One of the motifs:

[0661] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0662] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0663] Another motif:

[0664] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0665] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16),

[0666] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0667] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 82. An example of such a mutant IL-2 is mutant 2-153.

[0668] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0669] One of the motifs:

[0670] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0671] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0672] Another motif:

[0673] - Inserted by inserting a Q residue (i.e., containing a Q100 residue, preferably obtained via an E100Q mutation) at the E100 position of reference SEQ ID NO: 1, and

[0674] - Contains or consists of the sequence GSQ, preferably contains or consists of the sequence GSQST (SEQ ID NO: 18),

[0675] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0676] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 83. An example of such a mutant IL-2 is mutant 2-154.

[0677] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0678] One of the motifs:

[0679] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0680] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0681] Another motif:

[0682] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) (i.e., the N-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSPGAPTSSSTKKTQ).

[0683] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0684] In one embodiment, a sequence containing or composed of LQS, preferably containing or composed of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or composed of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0685] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 84. An example of such a mutant IL-2 is mutant 2-155.

[0686] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0687] One of the motifs:

[0688] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0689] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0690] Another motif:

[0691] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) (i.e., the N-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42)).

[0692] Optionally, the mutant IL-2 comprises replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0693] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or composed of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0694] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 85. An example of such a mutant IL-2 is mutant 2-156.

[0695] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0696] One of the motifs:

[0697] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0698] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0699] Another motif:

[0700] - Inserted by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing or consisting of TQG (SEQ ID NO: 44) (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing or consisting of TQG (SEQ ID NO: 44)).

[0701] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0702] In one embodiment, a sequence containing TQG or consisting of TQG, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or consisting of TQGASSTKKTQ (SEQ ID NO: 44), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0703] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 86. An example of such a mutant IL-2 is mutant 2-157.

[0704] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0705] One of the motifs:

[0706] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0707] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0708] Another motif:

[0709] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GGLQSP (SEQ ID NO: 47)).

[0710] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0711] In one embodiment, a sequence containing or composed of LQS, preferably containing or composed of GGLQSP (SEQ ID NO: 47) or composed of GGLQSP (SEQ ID NO: 47), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0712] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 87. An example of such a mutant IL-2 is mutant 2-158.

[0713] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0714] One of the motifs:

[0715] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0716] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0717] Another motif:

[0718] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GLQSP (SEQ ID NO: 48) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GLQSP (SEQ ID NO: 48)).

[0719] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0720] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing GLQSP (SEQ ID NO: 48) or composed of GLQSP (SEQ ID NO: 48), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0721] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 88. An example of such a mutant IL-2 is mutant 2-159.

[0722] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0723] One of the motifs:

[0724] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0725] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0726] Another motif:

[0727] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of LQSP (SEQ ID NO: 49) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSP (SEQ ID NO: 49)).

[0728] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0729] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSP (SEQ ID NO: 49) or composed of LQSP (SEQ ID NO: 49), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0730] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 89. An example of such a mutant IL-2 is mutant 2-160.

[0731] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0732] One of the motifs:

[0733] - Inserted by inserting a Q residue (i.e., containing a Q42 residue, preferably obtained through an F42Q mutation) at the F42 position of reference SEQ ID NO: 1, and

[0734] - Contains or consists of the sequence LTQR (SEQ ID NO: 2), preferably contains or consists of the sequence MLTQRF (SEQ ID NO: 12), and

[0735] Another motif:

[0736] - Insertion is achieved by inserting a sequence containing or consisting of QGA at the C end (i.e., the C end contains a sequence containing or consisting of QGA).

[0737] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0738] In one embodiment, a sequence containing or composed of QGAs is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0739] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 90. An example of such a mutant IL-2 is mutant 2-161.

[0740] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least three non-endogenous transglutaminase motifs.

[0741] One of the motifs:

[0742] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0743] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0744] Another motif:

[0745] - Inserted by inserting a Q residue (i.e., containing a Q51 residue, preferably obtained via a T51Q mutation) at the T51 position of reference SEQ ID NO: 1, and

[0746] - Contains or consists of the sequence AQA, preferably contains or consists of the sequence LAQALK (SEQ ID NO: 106), and

[0747] Another motif:

[0748] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0749] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16),

[0750] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0751] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 119. An example of such a mutant IL-2 is mutant 2-127.

[0752] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least three non-endogenous transglutaminase motifs.

[0753] One of the motifs:

[0754] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0755] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0756] Another motif:

[0757] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0758] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16), and

[0759] Another motif:

[0760] - Insertion is achieved by inserting a Q residue (i.e., containing a Q110 residue, preferably obtained via an E110Q mutation) at the E110 position of reference SEQ ID NO: 1, and

[0761] - Contains or consists of the sequence ALQ, preferably contains or consists of the sequence DYALQ (SEQ ID NO: 109).

[0762] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0763] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 120. An example of such a mutant IL-2 is mutant 2-128.

[0764] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0765] One of the motifs:

[0766] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0767] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0768] Another motif:

[0769] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GGLQSP (SEQ ID NO: 47)).

[0770] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0771] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 123. An example of such a mutant IL-2 is mutant 2-148.

[0772] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0773] One of the motifs:

[0774] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0775] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQAKF (SEQ ID NO: 11), and

[0776] Another motif:

[0777] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GLQSP (SEQ ID NO: 48) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GLQSP (SEQ ID NO: 48)).

[0778] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0779] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing GLQSP (SEQ ID NO: 48) or composed of GLQSP (SEQ ID NO: 48), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0780] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 124. An example of such a mutant IL-2 is mutant 2-149.

[0781] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, mutant IL-2 derived from human IL-2 having SEQ ID NO: 1 or a variant thereof contains at least two non-endogenous transglutaminase motifs, one of which is:

[0782] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0783] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10).

[0784] In one implementation, another non-endogenous transglutaminase motif is defined as described above.

[0785] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0786] One of the motifs:

[0787] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0788] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0789] Another motif:

[0790] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0791] - Contains or consists of the sequence LFQ, preferably contains or consists of the sequence KLFQLRPRD (SEQ ID NO: 15),

[0792] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0793] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 91. An example of such a mutant IL-2 is mutant 2-162.

[0794] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0795] One of the motifs:

[0796] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0797] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0798] Another motif:

[0799] - Inserted by inserting a Q residue (i.e., containing a Q79 residue, preferably obtained via an H79Q mutation) at the H79 position of reference SEQ ID NO: 1, and

[0800] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence KLLQLRPRD (SEQ ID NO: 16),

[0801] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0802] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 92. An example of such a mutant IL-2 is mutant 2-163.

[0803] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0804] One of the motifs:

[0805] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0806] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0807] Another motif:

[0808] - Inserted by inserting a Q residue (i.e., containing a Q100 residue, preferably obtained via an E100Q mutation) at the E100 position of reference SEQ ID NO: 1, and

[0809] - Contains or consists of the sequence GSQ, preferably contains or consists of the sequence GSQST (SEQ ID NO: 18),

[0810] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0811] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 93. An example of such a mutant IL-2 is mutant 2-164.

[0812] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0813] One of the motifs:

[0814] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0815] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0816] Another motif:

[0817] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) (i.e., the N-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42) or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42)).

[0818] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0819] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSPTSSSTKKTQ (SEQ ID NO: 42) or composed of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0820] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 94. An example of such a mutant IL-2 is mutant 2-166.

[0821] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0822] One of the motifs:

[0823] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0824] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0825] Another motif:

[0826] - Inserted by inserting a sequence containing or consisting of TQG at the N-terminus, preferably containing or consisting of TQG (SEQ ID NO: 44) (i.e., containing a sequence containing or consisting of TQG at the N-terminus, preferably containing or consisting of TQG (SEQ ID NO: 44)).

[0827] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0828] In one embodiment, a sequence containing TQG or consisting of TQG, preferably containing TQGASSTKKTQ (SEQ ID NO: 44) or consisting of TQGASSTKKTQ (SEQ ID NO: 44), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0829] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 95. An example of such a mutant IL-2 is mutant 2-167.

[0830] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0831] One of the motifs:

[0832] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0833] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0834] Another motif:

[0835] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of LQSP (SEQ ID NO: 49) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of LQSP (SEQ ID NO: 49)).

[0836] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0837] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing LQSP (SEQ ID NO: 49) or composed of LQSP (SEQ ID NO: 49), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0838] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 96. An example of such a mutant IL-2 is mutant 2-170.

[0839] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0840] One of the motifs:

[0841] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0842] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0843] Another motif:

[0844] - Insertion is achieved by inserting a sequence containing or consisting of QGA at the C end (i.e., the C end contains a sequence containing or consisting of QGA).

[0845] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0846] In one embodiment, a sequence containing or composed of QGAs is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0847] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 97. An example of such a mutant IL-2 is mutant 2-171.

[0848] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0849] One of the motifs:

[0850] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0851] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQFKF (SEQ ID NO: 10) or consists of the sequence LLQFKF (SEQ ID NO: 10), and

[0852] Another motif:

[0853] - Inserted by inserting a Q residue (i.e., containing a Q51 residue, preferably obtained via a T51Q mutation) at the T51 position of reference SEQ ID NO: 1, and

[0854] - Contains or consists of the sequence AQA, preferably contains or consists of the sequence LAQALK (SEQ ID NO: 106).

[0855] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0856] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 121. An example of such a mutant IL-2 is mutant 2-129.

[0857] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0858] One of the motifs:

[0859] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0860] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQ, preferably LLQFKF (SEQ ID NO: 10),

[0861] Another motif:

[0862] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the N-terminus, preferably containing the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or consisting of the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) (i.e., the N-terminus contains a sequence containing or consisting of LQS, preferably containing LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or consisting of the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40)).

[0863] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0864] In one embodiment, a sequence containing or composed of LQS, preferably containing or composed of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40) or composed of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), is inserted at the N-terminus of the sequence having SEQ ID NO: 50.

[0865] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 125. An example of such a mutant IL-2 is mutant 2-165.

[0866] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0867] One of the motifs:

[0868] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0869] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQ, preferably LLQFKF (SEQ ID NO: 10),

[0870] Another motif:

[0871] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GGLQSP (SEQ ID NO: 47) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GGLQSP (SEQ ID NO: 47)).

[0872] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0873] In one embodiment, a sequence containing or composed of LQS, preferably containing or composed of GGLQSP (SEQ ID NO: 47) or composed of GGLQSP (SEQ ID NO: 47), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0874] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 126. An example of such a mutant IL-2 is mutant 2-168.

[0875] In one embodiment, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least two non-endogenous transglutaminase motifs.

[0876] One of the motifs:

[0877] - Inserted by inserting a Q residue (i.e., containing a Q41 residue, preferably obtained via a T41Q mutation) at the T41 position of reference SEQ ID NO: 1, and

[0878] - Contains or consists of the sequence LLQ, preferably contains or consists of the sequence LLQ, preferably LLQFKF (SEQ ID NO: 10),

[0879] Another motif:

[0880] - Insertion is achieved by inserting a sequence containing or consisting of LQS at the C-terminus, preferably containing or consisting of GLQSP (SEQ ID NO: 48) (i.e., the C-terminus contains a sequence containing or consisting of LQS, preferably containing or consisting of GLQSP (SEQ ID NO: 48)).

[0881] Optionally, wherein with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with a glutamate residue (i.e., the Q74E mutation); or (iii) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

[0882] In one embodiment, a sequence containing LQS or composed of LQS, preferably containing GLQSP (SEQ ID NO: 48) or composed of GLQSP (SEQ ID NO: 48), is inserted at the C-terminus of the sequence having SEQ ID NO: 1.

[0883] In one embodiment, the mutant IL-2 comprises or consists of a sequence having SEQ ID NO: 127. An example of such a mutant IL-2 is mutant 2-169.

[0884] The present invention also relates to a method for incorporating at least one non-endogenous transglutaminase motif into human IL-2 having the sequence SEQ ID NO: 1 or a variant thereof, the method comprising the steps of:

[0885] - Insert Q residues at one or more of the following positions in reference SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130, and / or

[0886] - Insert a motif at the N-terminus or C-terminus, wherein the motif comprises a sequence selected from groups containing LQS and TQG or groups consisting of LQS and TQG.

[0887] Optionally, the method further includes the following steps:

[0888] (i) Replace the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation).

[0889] (ii) Replace the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation), or

[0890] (iii) The cysteine ​​residue at position 125 is replaced with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation), and the glutamine residue at position 74 is replaced with any other amino acid residue, preferably a glutamic acid residue (i.e., the Q74E mutation).

[0891] In one embodiment, the method further includes, with reference to SEQ ID NO: 1, the step of replacing the phenylalanine residue at position 42 with any other amino acid residue, preferably an alanine residue (i.e., the F42A mutation).

[0892] In one embodiment, the method includes inserting at least two non-endogenous transglutaminase motifs.

[0893] In one embodiment, referring to SEQ ID NO: 1, one of the non-endogenous transglutaminase motifs includes the insertion of a Q residue at the T41 or F42 position.

[0894] The implementation scheme for mutant IL-2 described herein is adapted to the method described herein with the necessary modifications.

[0895] The present invention also relates to fusion proteins comprising mutant IL-2 as defined above.

[0896] In one embodiment, the fusion protein includes at least one masking portion, wherein the at least one masking portion is linked to at least one Q residue of a non-endogenous transglutaminase motif of the mutant IL-2 as defined above.

[0897] In one embodiment, the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs, and the fusion protein comprises at least two masking portions, wherein the at least two masking portions are linked to the Q residue of each non-endogenous transglutaminase motif of the mutant IL-2 as defined above.

[0898] In one embodiment, when the fusion protein comprises at least two masking portions, the at least two masking portions are different. In another embodiment, when the fusion protein comprises at least two masking portions, the at least two masking portions are similar.

[0899] The masking portion used in this article refers to the portion that, when linked to the Q residue of the non-endogenous transglutaminase motif of mutant IL-2, prevents or reduces the signal transduction of mutant IL-2, or the binding or affinity of mutant IL-2 to its receptor.

[0900] Methods for measuring IL-2 signaling are well known in the art and include, for example, measuring the induction of IL-2 receptor signaling (e.g., by detecting phosphorylated STAT5a), measuring the induction of T cell proliferation (e.g., by using CellTrace, in particular). TM The Ki-67 cell proliferation assay directly assesses T cell proliferation in the presence of IL-2 in T cell activation assays (including, for example, activation of cells with CD3 and CD28 in the presence of IL-2, or using IL-2-dependent cell lines, such as the CTLL2 cell line) and / or measures the upregulation of activation markers (such as CD25, CD69, cytotoxic molecules such as granzyme B, etc.).

[0901] Protein affinity is usually expressed as the equilibrium dissociation constant (K). D Therefore, in one implementation, surface plasmon resonance (SPR, for example, using BIAcore) is employed. ® Determine the binding kinetics of mutant IL-2 to its receptor, such as K. D .

[0902] Other methods for measuring protein binding or affinity are well known in the art and include, for example, cell binding assays, such as competitive binding assays.

[0903] In one embodiment, the mutant IL-2 contained in the fusion protein as defined above exhibits reduced binding or affinity to its receptor compared to free (i.e., not binding to the masking portion) mutant IL-2. In one embodiment, the mutant IL-2 contained in the fusion protein as defined above exhibits reduced binding or affinity to its receptor by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to free (i.e., not binding to the masking portion) mutant IL-2.

[0904] In one embodiment, the mutant IL-2 contained in the fusion protein as defined above exhibits reduced binding or affinity for the IL-2Rα, β, and / or γ chains compared to free (i.e., not binding to the masking portion) mutant IL-2. In one embodiment, the mutant IL-2 contained in the fusion protein as defined above exhibits reduced binding or affinity for the IL-2Rα, β, and / or γ chains by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to free (i.e., not binding to the masking portion) mutant IL-2.

[0905] In one embodiment, the mutant IL-2 contained in the fusion protein as defined above exhibits reduced binding or affinity to low-, intermediate-, and / or high-affinity IL-2 receptors compared to free (i.e., not binding to the masking portion) mutant IL-2. In one embodiment, the mutant IL-2 contained in the fusion protein as defined above exhibits reduced binding or affinity to low-, intermediate-, and / or high-affinity IL-2 receptors by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to free (i.e., not binding to the masking portion) mutant IL-2.

[0906] As disclosed herein, at least one of the masking components can be any type of molecule.

[0907] In one embodiment, at least one masking component is selected from the group consisting of or composed of the following: polysaccharides, lipids, proteins, peptides, small organic molecules, and polymers.

[0908] In one implementation, at least one masking component is a polysaccharide.

[0909] In one embodiment, at least one of the masking components is a polymer, preferably a water-soluble polymer.

[0910] In one embodiment, the water-soluble polymer is selected from the group consisting of or comprising: polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, poly... Azoline (POZ), poly(N-acryloylmorpholine), and combinations thereof.

[0911] In one embodiment, the water-soluble polymer is polyethylene glycol (PEG).

[0912] In one embodiment, at least one masking portion is a straight-chain PEG. As used herein, a straight-chain PEG refers to a PEG consisting of a single straight chain.

[0913] In one embodiment, at least one masking portion is a linear PEG of up to about 20 kDa, preferably about 10 kDa.

[0914] In one embodiment, at least one masking component is a branched PEG. As used herein, a branched PEG refers to a PEG consisting of at least two straight-chain PEGs linked together. An example of a branched PEG is a Y-shaped PEG, such as a Y-shaped PEG having two PEGs of approximately 20 kDa.

[0915] In one embodiment, at least one masking portion is a PEG with a weight of about 5 to about 50 kDa, preferably about 10 to about 40 kDa.

[0916] It should be understood that when a fusion protein contains at least two masking portions, the at least two masking portions can be similar or different PEGs.

[0917] In one embodiment, the fusion protein further comprises at least one binding moiety, such as an antibody or a fragment thereof, or an antibody mimic. As used herein, a binding moiety refers to any molecule capable of binding to a target.

[0918] In one implementation, the binding portion and the masking portion are a single molecule, meaning that the single molecule can serve as both the masking portion and the binding portion.

[0919] In one embodiment, the fusion protein further comprises at least one antibody or an antigen-binding fragment thereof.

[0920] In one embodiment, at least one antibody or its antigen-binding fragment contained in the fusion protein is selected from the group consisting of or composed of: whole antibodies, single-chain antibodies, dimer single-chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, bispecific antibodies, biantibodies, triantibodies, tetraantibodies, nanobodies, domain antibodies, and unibody, preferably at least one antibody or antigen-binding fragment is Fab.

[0921] In one embodiment, at least one antibody or its antigen-binding fragment is polyclonal. In another embodiment, at least one antibody or its antigen-binding fragment is monoclonal.

[0922] In one embodiment, at least one antibody or its antigen-binding fragment is humanized. In another embodiment, at least one antibody or its antigen-binding fragment contained in the fusion protein is a fully or substantially human antibody or fragment thereof.

[0923] It should be understood that antibodies or antigen-binding fragments thereof contained in the fusion protein according to the invention may be modified using known methods.

[0924] Framework residues within the heavy chain variable region (VH) and / or light chain variable region (VL) can also be modified, for example, to improve the properties of the antibody or fragment. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody or fragment. For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence.

[0925] As another example, an antibody or its antigen-binding fragment may be deglycosylated (i.e., the antibody or fragment lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody or fragment for the antigen, or to change the antibody-dependent cytotoxic activity of the antibody or its fragment. Such carbohydrate modification can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence.

[0926] In another embodiment, the fusion protein further comprises at least one antibody mimic. Some examples of antibody mimics include, but are not limited to, affibody, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, fynomer, and versabody.

[0927] In one embodiment, at least one antibody or antigen-binding fragment or at least one antibody mimic targets an antigen, such as a cancer antigen or an immune checkpoint protein.

[0928] Examples of cancer antigens include, but are not limited to, Fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), nerve growth factor receptor (NGFR), insulin-like growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGFR), or hormone receptor (melanocortin 1 receptor (MC 1R, MSHR)).

[0929] Examples of immune checkpoint proteins include, but are not limited to,

[0930] In one embodiment, at least one antibody or antigen-binding fragment or antibody mimic targets HER2. HER2 (human epidermal growth factor receptor 2, also known as CD340 (differentiation cluster 340)) as used herein is a protein encoded by the ERBB2 gene.

[0931] In one embodiment, at least one antibody or antigen-binding fragment or antibody mimic targets PD-1. PD-1 (programmed cell death protein 1, also known as CD279 (differentiation cluster 279)) as used herein is a protein encoded by the PDCD1 gene.

[0932] In one embodiment, the fusion protein comprises at least one mutant IL-2, at least one masking portion and at least one binding portion, preferably an antibody or an antigen-binding fragment thereof or an antibody mimic, wherein at least one masking portion is linked to at least one mutant IL-2 via a connector, and wherein at least one mutant IL-2 is linked to at least one binding portion, preferably an antibody or an antigen-binding fragment thereof or an antibody mimic, via a further connector.

[0933] In one embodiment, the linker connecting (i) at least one masking portion to at least one mutant IL-2 and (ii) at least one mutant IL-2 to at least one binding portion (preferably an antibody or its antigen-binding fragment or antibody mimic) is similar. In another embodiment, the linker connecting (i) at least one masking portion to at least one mutant IL-2 and (ii) at least one mutant IL-2 to at least one binding portion (preferably an antibody or its antigen-binding fragment or antibody mimic) is different.

[0934] In one embodiment, the fusion protein comprises at least one mutant IL-2, at least two masking portions and at least one binding portion, preferably an antibody or its antigen-binding fragment or antibody mimic, wherein at least two masking portions are linked to at least one mutant IL-2 via a linker.

[0935] In one embodiment, the connectors linking at least two masking portions to at least one mutant IL-2 are similar. In another embodiment, the connectors linking at least two masking portions to at least one mutant IL-2 are different.

[0936] In one embodiment, the adapter linking at least one mutant IL-2 to at least one binding moiety (preferably an antibody or its antigen-binding fragment or antibody mimic) is a cleavable adapter. In another embodiment, the adapter linking at least one mutant IL-2 to at least one binding moiety (preferably an antibody or its antigen-binding fragment or antibody mimic) is a non-cleavable adapter. It should be understood that the nature of the adapter (i.e., cleavable or non-cleavable) will depend on the therapeutic application, as depending on the conditions, it may be desirable to have releasable IL-2 or IL-2 irreversibly linked to the binding moiety (preferably an antibody or its antigen-binding fragment or antibody mimic).

[0937] In one embodiment, the connector linking at least one masking portion to at least one mutant IL-2 is a cleavable connector. In another embodiment, the connector linking at least one masking portion to at least one mutant IL-2 is a non-cleavable connector. It should be understood that the nature of the connector (i.e., cleavable or non-cleavable) will depend on the therapeutic application, as depending on the conditions, it may be desirable to have a releasable masking portion or an IL-2 irreversibly linked to the masking portion.

[0938] In one embodiment, the fusion protein comprises at least one or at least two masking portions, at least one mutant IL-2, and at least one binding portion, preferably an antibody or its antigen-binding fragment or antibody mimic, wherein the elements are connected by a cleavable adapter.

[0939] In one embodiment, the fusion protein comprises at least one or at least two masking portions, at least one mutant IL-2, and at least one binding portion, preferably an antibody or its antigen-binding fragment or antibody mimic, wherein the elements are connected by an incleavable linker.

[0940] In one embodiment, the fusion protein comprises at least one or at least two masking portions, at least one mutant IL-2, and at least one binding portion, preferably an antibody or its antigen-binding fragment or antibody mimic, wherein at least one or at least two masking portions are linked to at least one mutant IL-2 via an uncleavable linker, and wherein at least one binding portion (preferably an antibody or its antigen-binding fragment or antibody mimic) is linked to at least one mutant IL-2 via a cleavable linker.

[0941] In one embodiment, the fusion protein comprises at least one or at least two masking portions, at least one mutant IL-2, and at least one binding portion, preferably an antibody or an antigen-binding fragment thereof or an antibody mimic, wherein at least one or at least two masking portions are linked to at least one mutant IL-2 via a cleavable linker, and wherein at least one binding portion (preferably an antibody or an antigen-binding fragment thereof or an antibody mimic) is linked to at least one mutant IL-2 via an uncleavable linker.

[0942] In one implementation, the uncuttable linker is a peptide with a length of 2 to 10 amino acids.

[0943] For example, glycine-serine duals provide a particularly suitable hinge domain (GS connector). In one embodiment, the hinge domain is a Gly / Ser connector. Some examples of Gly / Ser connectors include, but are not limited to, GS connectors, G2S connectors, G3S connectors, and G4S connectors.

[0944] Examples of G2S connectors include, but are not limited to, GGS.

[0945] The G3S linker contains the amino acid sequence (Gly-Gly-Gly-Ser). n Also known as (GGGS) n (SEQ ID NO: 100), where n is a positive integer equal to or greater than 1 (e.g., n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 or n=10).

[0946] The G4S linker contains the amino acid sequence (Gly-Gly-Gly-Gly-Ser). n Also known as (GGGGS) n(SEQ ID NO:101), where n is a positive integer equal to or greater than 1 (e.g., n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 or n=10).

[0947] As another example, connector A4T (SEQ ID NO: 129) can also be used in the context of this invention.

[0948] In one embodiment, the cleavable linker is a cleavable peptide with a length of 2 to 10 amino acids.

[0949] In one implementation, the cuttable connector can be cut by enzymes, light irradiation, pH, or chemical reagents.

[0950] In one embodiment, the cleavable adapter can be cleaved by an enzyme, such as an adapter that can be cleaved by a protease, an adapter that can be cleaved by a β-glucuronidase, or a peptide adapter.

[0951] In one embodiment, the cuttable connector can be pH-cut. Some examples of pH-cuttable connectors include, but are not limited to, hydrazine connectors, maleic amide connectors, or cis-aconityl connectors.

[0952] An example of the fusion protein of the present invention is in Figure 14 The fusion protein of the present invention may or may not have a binding moiety, and may or may not have a linker between the binding moiety and the IL-2 mutant. Regarding the linker, the linker between the binding moiety and the IL-2 mutant may or may not be cleavable by proteolysis, and the linker between the masking moiety and IL-2 may or may not be cleavable.

[0953] Another object of the present invention is the isolation of nucleic acids that encode mutant IL-2 or fusion proteins according to the present invention.

[0954] As used in this article, "isolated nucleic acid" refers to nucleic acid that is substantially separated from other genomic DNA sequences and naturally occurring proteins or complexes (e.g., ribosomes and polymerases) that accompany the natural sequence. The term encompasses nucleic acid sequences that have been removed from their natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized from heterologous systems. Essentially pure nucleic acid includes the isolated form of nucleic acid. This, of course, refers to the initially isolated nucleic acid and does not exclude genes or sequences subsequently added artificially to the isolated nucleic acid.

[0955] Typically, the nucleic acid is a DNA or RNA molecule that can be contained in any suitable vector, such as a plasmid, granule, episome, artificial chromosome, bacteriophage, or viral vector.

[0956] Therefore, another object of the present invention is an expression vector containing nucleic acid encoding a mutant IL-2 or fusion protein according to the present invention.

[0957] The terms "vector," "cloning vector," and "expression vector" refer to a vector that introduces a DNA or RNA sequence (e.g., a foreign gene) into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence. Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to induce or direct the expression of the fusion protein after administration to the host. Some examples of promoters and enhancers for animal cell expression vectors include the early promoter and enhancer of SV40, the LTR promoter and enhancer of Moloney mouse leukemia virus, and the promoter and enhancer of immunoglobulin H chain. Any expression vector for animal cells can be used, as long as the gene encoding the fusion protein can be inserted and expressed. Some examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1β d2-4, etc. Other examples of plasmids include replication plasmids containing the origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenoviruses, retroviruses, herpesviruses, and AAV vectors. Such recombinant viruses can be produced using techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Some typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, Gpenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found in the art.

[0958] Another object of the present invention is an isolated host cell containing the vector. The host cell can be used for recombinant production of mutant IL-2 or fusion protein according to the present invention.

[0959] In one embodiment, the host cell may be a prokaryotic, yeast, or eukaryotic cell, preferably a mammalian cell, such as: monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells for subcloning in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL8287 or NSO (HPA culture preservation no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2), as well as the DSM PERC-6 cell line. Expression vectors suitable for each of these host cells are also well known in the art. It should be noted that the term "host cell" generally refers to a cultured cell line. In one embodiment, a whole human being in which an expression vector encoding the fusion protein according to the invention has been introduced is excluded from the definition of "host cell".

[0960] Another object of the present invention is a composition comprising, substantially comprising, or consisting of at least one mutant IL-2 according to the present invention, at least one fusion protein, at least one nucleic acid, or at least one expression vector.

[0961] As used herein, the phrase “consisting substantially of…” means that at least one mutant IL-2, fusion protein, nucleic acid, or expression vector is the only therapeutic agent or biologically active agent in the composition.

[0962] Another object of the present invention is a pharmaceutical composition comprising, substantially comprising, or comprising of: at least one mutant IL-2 according to the present invention, at least one fusion protein, at least one nucleic acid or at least one expression vector, and at least one pharmaceutically acceptable excipient.

[0963] The term "pharmaceutical-grade excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents. The excipient does not produce adverse, allergic, or other adverse reactions when administered to animals (preferably mammals, more preferably humans). For human use, the formulation should meet the sterility, pyrogenicity, and general safety and purity standards required by regulatory agencies (e.g., the FDA office or EMA).

[0964] Examples of pharmaceutically acceptable excipients that can be used in the compositions of the present invention include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acid metaglycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0965] In one embodiment, the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable carrier of the formulation capable of being injected into a subject. These may be, in particular, isotonic, sterile saline solutions (monophosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of these salts), or dry, particularly lyophilized compositions, which, depending on the circumstances, may form an injectable solution after the addition of sterile water or physiological saline.

[0966] Another object of the present invention is the use of mutant IL-2, fusion protein, nucleic acid or expression vector according to the invention as a drug.

[0967] Another object of the present invention is a medicine comprising, substantially comprising, or comprising of: at least one mutant IL-2 according to the present invention, at least one fusion protein, at least one nucleic acid, or at least one expression vector.

[0968] Another object of the present invention is the use of at least one mutant IL-2, at least one fusion protein, at least one nucleic acid, or at least one expression vector in the preparation of a medicament for treating a disease, disorder, or symptom in a subject in need of such treatment.

[0969] The composition, pharmaceutical composition, or drug is formulated for administration to a subject.

[0970] In one embodiment, the composition, pharmaceutical composition, or drug according to the invention is administered (or is to be administered) parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir.

[0971] In one embodiment, the composition, pharmaceutical composition, or drug is administered (or to be administered) by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheath, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0972] Examples of forms suitable for injection include, but are not limited to, solutions such as sterile aqueous solutions; gels; dispersions; emulsions; suspensions; solid forms suitable for adding liquid prior to use to prepare solutions or suspensions, such as powders; liposomes, etc.

[0973] The compositions, pharmaceutical compositions, or sterile injectable forms of the present invention can be aqueous or oily suspensions. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils are commonly used as solvents or suspending media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (e.g., oleic acid) and their glycerol derivatives can be used to prepare injectable formulations, as can natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylene forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms (including emulsions and suspensions). Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, are typically used to prepare pharmaceutical solid, liquid, or other dosage forms. They may also be used for formulation purposes.

[0974] In one embodiment, the mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or drug according to the invention is administered to a subject in need at a therapeutically effective amount.

[0975] However, it should be understood that the total daily dose of the IL-2 mutant, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or drug according to the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disease being treated and its severity; the activity of the IL-2 mutant, fusion protein, nucleic acid, or expression vector used; the subject's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific IL-2 mutant, fusion protein, nucleic acid, or expression vector used; the duration of treatment; the drugs used in combination with or concurrently with the specific IL-2 mutant, fusion protein, nucleic acid, or expression vector used; and similar factors known in the medical field. For example, starting the dose of the compound at a level lower than required to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved is entirely within the skill of those skilled in the art. The total dose required for each treatment can be administered via multiple doses or as a single dose.

[0976] The present invention also relates to at least one mutant IL-2, fusion protein, nucleic acid or expression vector as described herein, which is used as a drug, i.e. for treating (or for treating) a disease, disorder or symptom in a subject in need of such treatment.

[0977] The present invention also relates to at least one mutant IL-2, fusion protein, nucleic acid or expression vector as described herein, for use in promoting Treg cell expansion in subjects in which such subjects may treat diseases, disorders or symptoms.

[0978] The present invention also relates to methods for treating diseases, disorders or symptoms in subjects in need of such treatment or for promoting the expansion of Treg cells in subjects in need of such treatment, comprising administering to the subject at least one mutant IL-2, fusion protein, nucleic acid or expression vector as described herein.

[0979] Examples of diseases that can be treated in this invention include, but are not limited to, cancer and inflammatory diseases.

[0980] In one implementation plan, the disease, disorder, or symptom to be treated is cancer.

[0981] The term "cancer" as used herein has its general meaning in the art and includes, but is not limited to, solid tumors and hematogenous tumors. The term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" also encompasses both primary and metastatic cancers.

[0982] Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells from the following: bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, endometrium, pancreas, or uterus.

[0983] In addition, cancers can be selected from the following non-restrictive list: malignant growths; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; pilomatal carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; mixed hepatocellular carcinomas and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; adenocarcinomas associated with familial adenomatous polyposis; solid carcinomas; malignant carcinoid tumors; bronchoalveolar adenocarcinomas; papillary adenocarcinomas; chromophobe carcinomas; acidophilic carcinomas; oxyphilic adenocarcinomas; basophilic carcinomas; clear cell adenocarcinomas; granular cell carcinomas; follicular adenocarcinomas; papillary and follicular adenocarcinomas; non-encapsulating sclerosing carcinomas. Carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin appendage carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullar carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant roblastoma; Sertoli cell carcinoma; Malignant Leydig cell tumor (tumor); malignant lipocytoma; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoma; superficial diffuse melanoma; malignant melanoma within a giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; acinar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma;Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Cortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pineal tumor; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibroblastoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroblastoma; Primitive neuroectodermis Neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Paragranuloma; Malignant lymphoma - small (lymphocytic); malignant diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other specific non-Hodgkin lymphomas; malignant histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0984] In one implementation plan, the disease, disorder, or symptom to be treated is an inflammatory disease.

[0985] Examples of inflammatory diseases include, but are not limited to: arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, gouty arthritis, gout, chronic polyarthritis, periarthritis of the shoulder, cervical spondylosis, lumbosacral arthritis, enteropathic arthritis, ankylosing spondylitis, asthma, dermatitis, psoriasis, scleroderma, polymyositis, dermatomyositis, juvenile dermatomyositis, primary biliary cirrhosis, fibrosis, cystic fibrosis, pulmonary fibrosis, cirrhosis, endocardial myocardial fibrosis, dediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, and renal fibrosis. Keloids, scleroderma, joint fibrosis, late post-transplant and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, pemphigus, pemphigus vulgaris, pemphigus herpes-like, proliferative pemphigus, IgA pemphigus, erythematous pemphigus, bullous pemphigoid, gestational pemphigoid, mucocutaneous dermatitis, nodular pemphigoid, linear IgA bullous dermatitis, bullous lichen planus, acquired epidermolysis bullosa, autoimmune diabetes, diabetic retinopathy, diabetic nephropathy, diabetic angiopathy, ocular inflammation, uveitis, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty, chronic obstructive pulmonary disease (COPD). Obstructive pulmonary disease (COPD), glomerulonephritis, Graves' disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina pectoris, small artery disease, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, systemic sclerosis, antiphospholipid syndrome, Sjoegren's syndrome, autoimmune hemolytic anemia, colitis, Crohn's disease, ulcerative colitis, inflammatory bowel disease (IBD), embolism, pulmonary embolism, arterial embolism, venous embolism, allergic inflammation, cardiovascular disease, graft-related diseases, and graft-versus-host disease. Disease (GVHD), conditions associated with graft rejection, chronic rejection and tissue or cell allogeneic or xenograft grafts, autoimmune diseases, post-traumatic degeneration, stroke, transplant rejection, allergic diseases and hypersensitivity reactions (e.g., allergic rhinitis, allergic eczema), skin diseases, inflammatory skin conditions, or any combination thereof.

[0986] In one embodiment, the mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or pharmaceutical alone according to the invention are used.

[0987] In another embodiment, the mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or drug according to the invention is used in combination with at least one additional therapeutic agent.

[0988] In one embodiment, for concurrent administration, at least one additional therapeutic agent is administered as a combination or as a separate composition with the mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or drug according to the invention.

[0989] Other examples of therapeutic agents include, but are not limited to: chemotherapy agents, targeted cancer therapy, radiation therapy, immunotherapy agents or anticancer immunogens, anticancer antibodies, cytotoxic agents, antiangiogenic agents, cell cycle control / apoptosis regulators, hormone regulators, tyrosine kinase inhibitors (TIK), vaccines, immune checkpoint inhibitors, CAR-T and CAR-NK cell therapy, CAR macrophage therapy, tumor-infiltrating lymphocyte (TIL) therapy, immune engaging biologics, steroids, JAK inhibitors, and other immunosuppressive and / or anti-inflammatory drugs, including corticosteroids, such as glucocorticoids.

[0990] In one embodiment, at least one additional therapeutic agent is a therapeutic agent for treating a specific disease, disorder, or symptom to be treated in this invention.

[0991] For example, in order to treat cancer, at least one additional therapeutic agent may be selected from the group including, but not limited to, the following: chemotherapeutic agents, targeted cancer therapies, radiotherapy, immunotherapeutic agents or anticancer immunogens, anticancer antibodies, cytotoxic agents, antiangiogenic agents, cell cycle control / apoptosis regulators, hormone regulators, tyrosine kinase inhibitors (TIK), vaccines, immune checkpoint inhibitors, CAR-T and CAR-NK cell therapies, CAR macrophage therapy, tumor-infiltrating lymphocyte (TIL) therapy, immune-conjugating biologics, and other immunosuppressive and / or anti-inflammatory drugs selected from corticosteroids, such as glucocorticoids.

[0992] For example, in order to treat inflammatory diseases, at least one additional therapeutic agent may be selected from the group including, but not limited to, anti-inflammatory drugs, steroids and JAK inhibitors. Attached Figure Description

[0993] Figure 1 is a table showing the following: for a fusion protein comprising a mutant IL-2 according to the invention fused with Fab targeting HER2, its production and purification (concentration in mg / mL, total mass after purification in mg, yield in mg / L, purity obtained by SDS-PAGE), its ability to bind to HER2, CD25 and CD122 (yes or no), its ability to activate CD4 T cells, Treg cells, CD8 T cells and NK cells (mean EC50 in pM), and its ability to perform bioconjugation (yes or no, NT = not tested). Figure 1A Results are shown for fusion proteins containing 2-102, 2-105 to 2-107, 2-112 to 2-116 and 2-120IL-2 mutants. Figure 1B Results of fusion proteins containing IL-2 mutants from 2-121 to 2-126, 2-130, and 2-131 are shown.

[0994] Figure 2 This is a combination of two graphs showing the percentage of pSTAT5 expression on Treg cells (A) and CD8 T cells (B) induced by the following fusion proteins: IL2-Fab (i.e., wild-type IL-2 fused with Fab targeting HER2, used as a control), and fusion proteins containing 2-114 or 2-115 IL-2 mutants, 10 kDa PEG fused with the non-endogenous transglutaminase motif of IL-2, and Fab targeting HER2 (i.e., fusion proteins 2-114-P10 and 2-115-P10, respectively).

[0995] Figure 3 This is a combination of four graphs showing the percentage of pSTAT5 expression induced on CD4 T cells, CD8 T cells, Treg cells, and NK cells by a fusion protein comprising a 2-120 IL-2 mutant, a 10 kDa PEG fused to a non-endogenous transglutaminase motif of IL-2, and a Fab (2-120-P10) targeting HER2. Solid lines represent 2-120, and dashed lines represent the 2-120-P10 construct.

[0996] Figure 4 This is a combination of four graphs showing the percentage of pSTAT5 expression induced on CD4 T cells, CD8 T cells, Treg cells, and NK cells by a fusion protein comprising a 2-121 IL-2 mutant, a 10 kDa PEG fused to a non-endogenous transglutaminase motif of IL-2, and a Fab (2-121-P10) targeting HER2. Solid lines represent 2-121, and dashed lines represent the 2-121-P10 construct.

[0997] Figure 5 This is a combination of four graphs showing the percentage of pSTAT5 expression induced on CD4 T cells, CD8 T cells, Treg cells, and NK cells by a fusion protein comprising a 2-122 IL-2 mutant, a 10 kDa PEG fused to a non-endogenous transglutaminase motif of IL-2, and a Fab (2-122-P10) targeting HER2. Solid lines represent 2-122, and dashed lines represent the 2-122-P10 construct.

[0998] Figure 6 This is a combination of four graphs showing the percentage of pSTAT5 expression induced on CD4 T cells, CD8 T cells, Treg cells, and NK cells by a fusion protein comprising a 2-123 IL-2 mutant, a 10 kDa PEG fused to a non-endogenous transglutaminase motif of IL-2, and a Fab (2-123-P10) targeting HER2. Solid lines represent 2-123, and dashed lines represent the 2-123-P10 construct.

[0999] Figure 7 This is a combination of four graphs showing the percentage of pSTAT5 expression induced on CD4 T cells, CD8 T cells, Treg cells, and NK cells by a fusion protein comprising a 2-124 IL-2 mutant, a 10 kDa PEG fused to a non-endogenous transglutaminase motif of IL-2, and a Fab (2-124-P10) targeting HER2. Solid lines represent 2-124, and dashed lines represent the 2-124-P10 construct.

[1000] Figure 8 is a combination of seven graphs showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by the fusion protein according to the present invention. % STAT5 is normalized against IL2-Fab. It illustrates the effects of IL2-Fab (control) and the following fusion proteins: fusion proteins containing the 2-114 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-114 and 2-114-P10, respectively) (A); fusion proteins containing the 2-115 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-115 and 2-115-P10, respectively) (B); fusion proteins containing the 2-120 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-120 and 2-120-P10, respectively) (C); fusion proteins containing the 2-121 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-121 and 2-121-P10, respectively) (D); and fusion proteins containing the 2-124 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (D). The fusion proteins are IL-2 mutants, HER2-targeting Fab proteins with or without 10 kDa PEG (2-124 and 2-124-P10, respectively) (E); fusion proteins containing 2-125 IL-2 mutants, HER2-targeting Fab proteins with or without 10 kDa PEG (2-125 and 2-125-P10, respectively) (F); and fusion proteins containing 2-126 IL-2 mutants, HER2-targeting Fab proteins with or without 10 kDa PEG (2-126 and 2-126-P10, respectively) (G). Unstimulated (Unstim.) indicates unactivated cells.

[1001] Figure 9 The following table shows the production and purification (concentration in mg / mL, total mass after purification in mg, yield in mg / L, purity obtained by SDS-PAGE) of fusion proteins comprising mutant IL-2 according to the invention (i.e., 2-132 to 2-135, 2-137, 2-138 and 2-140 IL-2 mutants) fused with Fab targeting HER2, their ability to bind to HER2, CD25 and CD122 (yes or no), their ability to activate CD4 T cells, Treg cells, CD8 T cells and NK cells (mean EC50 in pM), and their ability to perform bioconjugation (yes or no, NT = not tested).

[1002] Figure 10 is a table showing the following: for a fusion protein comprising a mutant IL-2 according to the invention fused with Fab targeting HER2, its production and purification (concentration in mg / mL, total mass after purification in mg, yield in mg / L, purity obtained by SDS-PAGE), its ability to bind to HER2, CD25 and CD122 (yes or no), its ability to activate CD4 T cells, Treg cells, CD8 T cells and NK cells (mean EC50 in pM), and its ability to perform bioconjugation (yes or no, NT = not tested). Figure 10A Results are shown for fusion proteins containing IL-2 mutants 2-142 to 2-147 and 2-150 to 2-153. Figure 10B Results of fusion proteins containing 2-154 to 2-161 IL-2 mutants are shown.

[1003] Figure 11 The following table shows the production and purification (concentration in mg / mL, total mass after purification in mg, yield in mg / L, purity obtained by SDS-PAGE) of fusion proteins comprising mutant IL-2 according to the invention (i.e., 2-162 to 2-164, 2-166, 2-167, 2-170 and 2-171 IL-2 mutants) fused with Fab targeting HER2, their ability to bind to HER2, CD25 and CD122 (yes or no), their ability to activate CD4 T cells, Treg cells, CD8 T cells and NK cells (mean EC50 in pM), and their ability to perform bioconjugation (yes or no, NT = not tested).

[1004] Figure 12 is a combination of three graphs showing the percentage of pSTAT5 expression induced in Treg cells by IL2-Fab (control) and by the following fusion proteins according to the invention: fusion proteins containing the 2-130 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-130-P10 and 2-130, respectively) (A); fusion proteins containing the 2-135 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-135-P10 and 2-135, respectively) (B); and fusion proteins containing the 2-137 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-137-P10 and 2-137, respectively) (C). Unstimulated (Unstim.) indicates unactivated cells.

[1005] Figure 13 is a combination of three graphs showing the percentage of pSTAT5 expression on CD8 T cells induced by IL2-Fab (control) and by the following fusion proteins according to the invention: fusion proteins containing the 2-130 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-130-P10 and 2-130, respectively) (A); fusion proteins containing the 2-135 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-135-P10 and 2-135, respectively) (B); and fusion proteins containing the 2-137 IL-2 mutant, HER2-targeting Fab, and with or without 10 kDa PEG (2-137-P10 and 2-137, respectively) (C). Unstimulated (Unstim.) indicates unactivated cells.

[1006] Figure 14 This is a schematic diagram illustrating an example of a fusion protein according to the present invention. The fusion protein of the present invention may or may not have a binding portion, and may or may not have a linker between the binding portion and the IL-2 mutant. Regarding the linker, the linker between the binding portion and the IL-2 mutant may or may not be cleavable by proteolysis, and the linker between the masking portion and the IL-2 mutant may or may not be cleavable.

[1007] Figure 15 is a combination of four graphs showing the percentage of pSTAT5 expression on CD4 T cells (A), Treg cells (B), CD8 T cells (C), and NK cells (D) induced by IL2-Fab (control) and by fusion proteins containing the 2-130 IL-2 mutant, HER2-targeting Fab, and with or without 40 kDa PEG (2-130-P40 and 2-130, respectively). Unstimulated (Unstim.) indicates unactivated cells.

[1008] Figure 16 is a combination of four graphs showing the percentage of pSTAT5 expression on CD4 T cells (A), Treg cells (B), CD8 T cells (C), and NK cells (D) induced by IL2-Fab (control) and by fusion proteins containing the 2-132 IL-2 mutant, HER2-targeting Fab, and with or without 40 kDa PEG (2-132-P40 and 2-132, respectively). Unstimulated cells represent unactivated cells.

[1009] Figure 17 is a combination of three graphs showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by IL2-Fab (control) and the following: fusion proteins containing the 2-135 IL-2 mutant, PD-1-targeting Fab, with or without a 40 kDa PEG (2-135-P40 and 2-135, respectively) (A); fusion proteins containing the 2-136 IL-2 mutant, PD-1-targeting Fab, with or without a 40 kDa PEG (2-136-P40 and 2-136, respectively) (B); and fusion proteins containing the 2-137 IL-2 mutant, PD-1-targeting Fab, with or without a 40 kDa PEG (2-137-P40 and 2-137, respectively). % STAT5 is normalized for IL2-Fab.

[1010] Figure 18 This graph shows the percentage of pSTAT5 expression induced by IL2-Fab (control) and by fusion proteins (2-210-P40 and 2-210, respectively) containing the 2-210 IL-2 mutant, targeting HER2, and with or without 40 kDa PEG. % STAT5 is normalized to IL2-Fab.

[1011] Figure 19 The figure shows an in vivo PK study, which was conducted using a control fusion protein (IL2-Fab with the F42 mutation (non-α IL2-Fab) or IL2-Fab without the F42 mutation (WT IL2-Fab)) or a fusion protein containing the 2-132 mutant, targeting PD-1, and having a PEG of 10 kDa or 40 kDa.

[1012] Figure 20 This graph shows the percentage of pSTAT5 expression induced by IL2-Fab (control) and by fusion proteins containing the 2-222 IL-2 mutant, targeting PD-1, and with or without 40 kDa PEG (2-222-P40 and 2-222, respectively). % STAT5 is normalized for IL2-Fab.

[1013] Figure 21This graph shows the percentage of pSTAT5 expression induced by IL2-Fab (control) and by fusion proteins (2-138-P40 and 2-138, respectively) containing the 2-138 IL-2 mutant, targeting PD-1, and with or without 40 kDa PEG. % STAT5 is normalized for IL2-Fab.

[1014] Example

[1015] The present invention will be further illustrated by the following examples.

[1016] Materials and methods

[1017] Expression and purification of immune cytokine constructs

[1018] DNA encoding the amino acid sequences of the listed constructs was synthesized and cloned into the mammalian transient expression vector plasmid pETE V2. All constructs were expressed using a CHO-based transient expression system, and the resulting cell culture supernatant containing antibodies was clarified by centrifugation and filtration. All constructs were purified from the cell culture supernatant by affinity chromatography. The purified antibodies were buffer-exchanged in phosphate-buffered saline solution. Antibody purity was determined by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by semi-preparative SEC. Antibody concentration was determined by measuring absorbance at 280 nm and calculating the theoretical extinction coefficient.

[1019] pSTAT5 activation assay on hPBMC

[1020] PBMCs were washed and counted into a complete RPMI in the absence of IL2. Cells were then loaded at 1 × 10⁻⁶. 6 cells / ml and 7×10 5Cells were seeded per well and incubated at 37°C for 1 hour. Dilutes of the target immunocytokines from 1000 nM to 0.1 pM were prepared in PBS (Table 8) to produce 10× concentrations for each sample. 5 μL / well of the 10× immunocytokines was added to the cells to achieve a final concentration range of 100 nM to 0.01 pM, and the cells were incubated at 37°C and 5% CO2 for 15 minutes. Membrane staining antibodies specific to each individual cell type were added to the corresponding wells: anti-CD25-FITC (Biolegend 3561606), anti-CD127-PE (ebio 12-1278-42), anti-CD8-ECD (BC-B737660), anti-CD4-PC7 (BC-B737660), anti-CD3-Pacific Blue (BC-B49204), and anti-CD56-BV510 (Biolegend 318340). To prepare the FACS plates, 8 μL of R1 fixative was incubated in the wells at RT for 10 min, followed by a mixture of 65 μL FBS and 145 μL BufferPerm reagent, and incubated at 37°C for 5 min. The plates were centrifuged at 400 g for 2 min, and then 50 μL / well of intercellular staining antibody (pSTAT5-AF647-FL6) was added. The plates were incubated at RT in the dark for 30 min, followed by a wash once with 150 μL R4 buffer. After washing, each sample was resuspended in 150 μL of BufferR4 and transferred to a microburette for reading on a Navios EX.

[1021] Receptor binding affinity assay

[1022] Receptor binding assays were performed using a capture assay setup. Anti-human IgG (catalog number BR100839) was conjugated to a CM5 chip (GE Healthcare) to approximately 9000 RU using standard amine chemistry. Human IL-2Rα-Fc at 1 μg / ml in HBS-P+ running buffer was captured to approximately 60 RU on flow cell 2. Human IL-2Rγ-Fc at 1 μg / ml in HBS-P+ running buffer was captured to approximately 120 RU on flow cell 1. Her2 binding in the Fab portion was evaluated in the same assay setup, where Her2-Fc was captured on the chip and the analytes were run on the surface. A 4-point 3-fold dilution series of each analyte (immunocytokine) was used for single-cycle kinetic analysis. For both IL-2Rα and IL-2Rγ, an analyte concentration range of 66.6 nM to 1800 nM was used, and for HER2 binding, a range of 2.5 nM to 66.6 nM was used. Between each run, the chip was regenerated with 3 MMgCl2. A single-cycle kinetic scheme was run on a Biacore T200, and 1:1 kinetic analysis or steady-state affinity measurement was performed using Biacore T200 evaluation software V 2.0.1 after dual-reference subtraction.

[1023] A similar protocol is used when testing both masked and unmasked immune cytokines.

[1024] pK determination

[1025] Nunc MaxiSorp was coated with 25 μL / well of 2 μg / ml anti-nivolumab Fab antibody (BioRad-HCA299 human Fab) protein in coating buffer (PBS 1×). The plate was sealed and incubated overnight at 4°C. The sample wells were blocked with PBS w / v 1% BSA for 1 hour at room temperature, followed by washing with 3× PBS-0.05% Tween-20. Serum samples collected from the test group were prepared and added to the plate, then incubated at room temperature for 2 hours, followed by washing with 3× PBS-0.05% Tween-20. The detection antibody and anti-κ light chain HRP antibody (Novus NB500-331H) were added to the plate, and the incubation and washing steps detailed above were performed. The detection substrate (TMB) was then added, and the optical absorbance was measured at 450 nm.

[1026] result

[1027] Example 1: IL-2 mutant with at least one non-endogenous transglutaminase motif

[1028] A fusion protein comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif and a Fab targeting HER2 was constructed, and various parameters were investigated: i) its ability to be generated and purified, ii) its ability to bind HER2, CD25, and CD122, iii) its ability to activate CD4 T cells, Treg cells, CD8 T cells, and NK cells, and iv) its ability to bioconjugate with a range of PEG motifs of different sizes and structures, including but not limited to 10 kDa linear and 40 kDa branched motifs. The results are presented in Figure 1.

[1029] All constructs containing mutant IL-2 with at least one non-endogenous transglutaminase motif i) can be robustly generated and purified, ii) bind to HER2 and CD122 (IL-2Rβ), iii) activate CD4 T cells, CD8 T cells, and NK cells, and iv) bioconjugate with PEG. All fusion proteins except those containing the 2-106, 2-107, 2-130, and 2-131 IL-2 mutants bind to CD25 (IL-2Rα), while fusion proteins containing the 2-106, 2-107, 2-130, and 2-131 IL-2 mutants do not bind to CD25 and therefore exhibit reduced potency in Treg cell activation.

[1030] Furthermore, fusion proteins were also developed comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif as described herein, a PEG fused to the IL-2 mutant via transglutamination using a linker, and a Fab targeting HER2. Their ability to activate Treg cells, NK cells, and CD8 T cells was evaluated. Figure 2 As shown, the presence of 10 kDa PEG fused with the 2-114 or 2-115 IL-2 mutants prevented CD8 T cell activation and had a low effect on Treg cell activation. The same results were obtained with the 2-125 and 2-126 mutants (data not shown).

[1031] Figure 3-7As shown, the presence of PEG fused to IL-2 mutants 2-120, 2-121, 2-122, 2-123, or 2-124 prevented activation of CD4 T cells and CD8 T cells, and had a lower effect on the activation of NK cells and Treg cells. Figure 8 further shows that the presence of PEG fused to IL-2 mutants 2-114, 2-115, 2-120, 2-121, 2-124, 2-125, or 2-126 affected NK cell activation to varying degrees, depending on the IL-2 mutant. Regarding the 2-130 IL-2 mutant, as shown in Figures 12, 13, and 15, the presence of 10 kDa PEG fused to this IL-2 mutant strongly prevented the activation of both CD8 T cells and Treg cells, and the presence of 40 kDa PEG prevented the activation of CD4 and CD8 T cells as well as Treg cells and NK cells.

[1032] Figure 18 The presence of 40 kDa PEG fused with the 2-210 IL-2 mutant was shown to affect NK cell activation.

[1033] Therefore, these data indicate that fusion proteins containing mutant IL-2 with PEG fused to a non-endogenous transglutaminase motif prevent the activation of CD4 T cells and CD8 T cells, and affect the activation of NK cells and Treg cells to varying degrees depending on the IL-2 mutant contained in the fusion protein.

[1034] In summary, these data demonstrate that fusing PEG as a masking component with an IL-2 mutant possessing at least one non-endogenous transglutaminase motif via transglutaminase action enables the blocking of IL-2 signaling on cells. This masking is reversible, achieved through the use of a cleavable linker between PEG and IL-2; under suitable conditions, this linker will be cleaved and induce the release of IL-2.

[1035] Example 2: IL-2 mutant with at least one non-endogenous transglutaminase motif and an F42A mutation

[1036] A fusion protein was constructed comprising a mutant IL-2 fused to a HER2-targeting Fab protein, possessing at least one non-endogenous transglutaminase motif and an F42A mutation. Various parameters were investigated: i) its ability to be produced and purified, ii) its ability to bind HER2, CD25, and CD122, iii) its ability to activate CD4 T cells, CD8 T cells, Treg cells, and NK cells, and iv) its ability to bioconjugate with PEG. Results were presented in... Figure 9 It is publicly available in China.

[1037] All constructs containing IL-2 mutants with at least one non-endogenous transglutaminase motif and an F42A mutation i) can be robustly generated and purified, ii) bind to HER2 and CD122 but not CD25, iii) activate CD4 T cells, CD8 T cells, Treg cells and NK cells, and iv) can be bioconjugated with PEG.

[1038] Furthermore, a fusion protein containing a mutant IL-2 with at least one non-endogenous transglutaminase motif and an F42A mutation, a PEG fused to the IL-2 mutant via transglutaminase action using a linker, and a Fab targeting HER2 was also developed, and its ability to activate Treg cells and CD8 T cells was evaluated. As shown in Figure 12, the presence of 10 kDa PEG fused to either the 2-135 or 2-137 IL-2 mutant prevented CD8 T cell activation and affected Treg cell activation to varying degrees depending on the IL-2 mutant.

[1039] Furthermore, as shown in Figure 16, the presence of 40 kDa PEG fused with the 2-132 mutant prevented the activation of CD4 T cells, CD8 T cells, Treg cells, and NK cells, with a low effect on the activation of Treg cells and NK cells.

[1040] Fusion proteins were also generated comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif and an F42A mutation, a 40 kDa PEG fused to the IL-2 mutant via transglutamination using an adapter, and a Fab targeting PD-1. Their effects on NK cell activation were evaluated. As shown in Figures 17, 20, and 21, the presence of the 40 kDa PEG fused to mutants 2-135, 2-136, 2-137, 2-138, or 2-222 affected NK cell activation.

[1041] In vivo PK studies were also conducted using such a fusion protein, which comprises a mutant IL-2 (i.e., the 2-132 mutant) having at least one non-endogenous transglutaminase motif and an F42A mutation, a 10 kDa or 40 kDa PEG fused to the IL-2 mutant via transglutamination using a linker, and a Fab targeting PD-1. Figure 19 As shown, the fusion protein with a branched 40kD PEG showed improved PK compared to the fusion protein with a straight-chain 10kD PEG. However, both masked fusion proteins showed improved PK compared to the control fusion protein.

[1042] Therefore, these data indicate that fusion proteins containing mutant IL-2 with PEG and F42A mutations fused to non-endogenous transglutaminase motifs prevent the activation of CD8 T and CD4 T cells and affect Treg and NK cell activation to varying degrees depending on the IL-2 mutant contained in the fusion protein.

[1043] Furthermore, these data indicate that the addition of PEG improves the PK of the fusion protein in vivo.

[1044] Example 3: IL-2 mutant with at least two non-endogenous transglutaminase motifs

[1045] A fusion protein containing a mutant IL-2 with at least two non-endogenous transglutaminase motifs fused to a HER2-targeting Fab protein was constructed, and various parameters were investigated: i) its ability to be produced and purified, ii) its ability to bind HER2, CD25, and CD122, and iii) its ability to activate CD4 T cells, Treg cells, and NK cells. Results were presented in... Figure 10A and 10B It is publicly available in China.

[1046] All constructs containing IL-2 mutants with at least two non-endogenous transglutaminase motifs i) can be robustly generated and purified, ii) bind to HER2 and CD122 but not CD25, and iii) can activate CD4 T cells, CD8 T cells, Treg cells and NK cells.

[1047] Therefore, these data suggest that two non-endogenous transglutaminase motifs can be combined to obtain functional IL-2 mutants and fusion proteins.

[1048] Example 4: A mutant IL-2 with at least two non-endogenous transglutaminase motifs, one of which It is the sequence LLQFKF (SEQ ID NO: 10) containing the Q41 residue.

[1049] A fusion protein containing a mutant IL-2 fused to a HER2-targeting Fab protein, comprising at least two non-endogenous transglutaminase motifs, one of which is the sequence LLQFKF (SEQ ID NO: 10) with a Q41 residue, was constructed. Various parameters were investigated: i) its ability to be produced and purified, ii) its ability to bind HER2, CD25, and CD122, iii) its ability to activate CD4 T cells, Treg cells, and NK cells, and iv) its ability to bioconjugate with PEG. Results were presented in... Figure 11 It is publicly available in China.

[1050] All constructs containing an IL-2 mutant having at least two non-endogenous transglutaminase motifs, one of which is the sequence LLQFKF (SEQ ID NO: 10) with Q41 residues, i) can be robustly generated and purified, ii) bind to HER2 and CD122 and CD25, iii) activate CD4 T cells, CD8 T cells and NK cells but not Treg cells, and iv) can be bioconjugated with PEG (in those tested).

[1051] Therefore, these data suggest that two non-endogenous transglutaminase motifs (one of which is the sequence LLQFKF (SEQ ID NO: 10) with Q41 residues) can be combined to obtain functional IL-2 mutants and fusion proteins.

Claims

1. A mutant IL-2 derived from human IL-2 having the sequence SEQ ID NO: 1 or a variant thereof, wherein, compared to SEQ ID NO: 1 or a variant thereof, the mutant IL-2 contains at least one non-endogenous transglutaminase motif, wherein said motif is inserted by means of: - Insert Q residues at one or more of the following positions in reference SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110, and S130, and / or - Insert a motif at the N-terminus or C-terminus, wherein the motif comprises a sequence selected from the group consisting of or selected from the group consisting of LQS and TQG. Optionally, with reference to SEQ ID NO: 1, the mutant IL-2 comprises (i) replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation); (ii) replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation); or (iii) both replacing the cysteine ​​residue at position 125 with any other amino acid residue, preferably a serine residue (i.e., the C125S mutation) and replacing the glutamine residue at position 74 with any other amino acid residue, preferably a glutamate residue (i.e., the Q74E mutation).

2. The mutant IL-2 according to claim 1, wherein the at least one non-endogenous transglutaminase motif is inserted by inserting a Q residue at one or more of the following positions in reference SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, S130.

3. The mutant IL-2 according to claim 1 or claim 2, wherein the at least one non-endogenous transglutaminase motif comprises or is composed of a sequence selected from the group consisting of or selected from the following: X1LQ, wherein X1 is A, L, H, I or V; NX2Q, wherein X2 is Y, H or F; YRQ, LTQ; AQX3, wherein X3 is A or E, TEQ, LFQ; GSQ and VIQ, preferably selected from the group consisting of or selected from the following: X1LQ, wherein X1 is L, H, I or V; NX2Q, wherein X2 is Y, H or F; YRQ, LTQR (SEQ ID NO: 2); LFQ; GSQ and VIQ.

4. The mutant IL-2 according to any one of claims 1 to 3, wherein the at least one non-endogenous transglutaminase motif comprises or is composed of the following: - Selected from groups containing the following or sequences selected from the following: Preferably selected from the group consisting of the following or from the sequence of the following: And / or - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the N-end, and / or - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the C-terminus.

5. The mutant IL-2 according to any one of claims 1 to 4, wherein the at least one non-endogenous transglutaminase motif is inserted by means of: - Insert Q residues at one or more of the following positions in reference SEQ ID NO: 1: N33, T41, F42, H79, E100, and / or - Insert a motif at the N-terminus or C-terminus, wherein the motif comprises or consists of a sequence selected from the group consisting of or selected from the group consisting of LQS and TQG.

6. The mutant IL-2 according to any one of claims 1 to 5, wherein the at least one non-endogenous transglutaminase motif comprises or is composed of the following: - Selected from groups containing the following or sequences selected from the following: And / or - Selected from the group consisting of the following or the sequence consisting of the following: LQS and TQG, preferably wherein the sequence is added at the C-terminus or the N-terminus.

7. The mutant IL-2 according to any one of claims 1 to 6, wherein the at least one non-endogenous transglutaminase motif comprises or is composed of the following: - Selected from groups containing the following or sequences selected from the following: And / or - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the N-end, and / or - Selected from groups containing the following or sequences selected from the following: Preferably, the sequence is added at the C-terminus.

8. The mutant IL-2 according to any one of claims 1 to 7, wherein the mutant comprises or consists of a sequence selected from the group consisting of or selected from the following: SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 19, SEQ ID NO: 21 to SEQ ID NO: 29, SEQ ID NO: 31 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63, SEQ ID NO: 110 to SEQ ID NO: 117, more preferably selected from the group consisting of or selected from the following: SEQ ID NO: 19 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63, and SEQ ID NO: 110 and 111.

9. The mutant IL-2 according to any one of claims 1 to 8, wherein the mutant comprises or consists of sequences selected from the group consisting of or selected from the group consisting of: SEQ ID NO: 26 to SEQ ID NO: 28, SEQ ID NO: 30 to SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55 and SEQ ID NO: 58 to SEQ ID NO: 63 and SEQ ID NO: 110 and 111.

10. The mutant IL-2 according to any one of claims 1 to 9, wherein the mutant IL-2 further comprises replacing the F42 residue of reference SEQ ID NO: 1 with any other amino acid, more preferably replacing the F42 residue with an alanine residue.

11. A fusion protein comprising a mutant IL-2 according to any one of claims 1 to 10, and further comprising at least one masking portion, wherein the at least one masking portion is linked via a cleavable linker to at least one Q residue of a non-endogenous transglutaminase motif of the mutant IL-2.

12. The fusion protein according to claim 11, wherein the masking portion is polyethylene glycol (PEG), preferably linear or branched PEG.

13. The fusion protein according to any one of claims 11 to 12, wherein the fusion protein further comprises an antibody or an antigen-binding fragment thereof or an antibody mimic, preferably wherein the antibody or an antigen-binding fragment thereof or an antibody mimic is linked to the mutant IL-2 via a linker.

14. The fusion protein according to any one of claims 11 to 13, which is used as a drug.

15. The fusion protein according to any one of claims 11 to 14, for the treatment of cancer or inflammatory diseases.

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