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WO2025040797A1 - Protéines variantes d'interleukine-2 qui facilitent la conjugaison chimique covalente et leurs utilisations - Google Patents

Protéines variantes d'interleukine-2 qui facilitent la conjugaison chimique covalente et leurs utilisations Download PDF

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Publication number
WO2025040797A1
WO2025040797A1 PCT/EP2024/073705 EP2024073705W WO2025040797A1 WO 2025040797 A1 WO2025040797 A1 WO 2025040797A1 EP 2024073705 W EP2024073705 W EP 2024073705W WO 2025040797 A1 WO2025040797 A1 WO 2025040797A1
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seq
residue
mutant
sequence
mutation
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William JJ FINLAY
Thibaut De Smedt
Sue TOWNSEND
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Depth Charge Ltd
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Depth Charge Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2

Definitions

  • the disclosure relates to mutational variants of the IL-2 protein that facilitate chemical conjugation and their potential use as treatments for cancer or inflammatory disease.
  • Interleukin-2 is an example of a cytokine protein that regulates the activities of white blood cells (e.g. T cells and NK cells) that are responsible for immunity, responses to microbial infections, and in discriminating between foreign ("non-self") and "self".
  • IL-2 is therefore a molecule of strong potential therapeutic interest in both pro-inflammatory (e.g. in cancer) and anti-inflammatory (auto-immune disease) settings.
  • IL-2 mediates its effects by binding to the IL-2 receptor complex, thereby stimulating the growth of immune cells such as helper, cytotoxic and regulatory T cells.
  • the IL-2 receptor complex (IL-2R) consists of three chains, termed alpha (CD25 high affinity, non-signaling), beta (CD122 moderate affinity, signaling) and gamma (CD132, low affinity, signaling). Heterodimerization of the P and y subunits of IL-2R is essential for signaling in T cells. IL-2 can therefore signal either via intermediate- affinity dimeric CD122/CD132 IL-2R (Kd ⁇ IO“ y M) or high-affinity trimeric CD25/CD 122/CD 132 IE- 2R (Kd ⁇ 10 -11 M) complexes.
  • alpha CD25 high affinity, non-signaling
  • beta CD122 moderate affinity, signaling
  • gamma CD132, low affinity, signaling
  • IL-2R complex Differential expression levels of these two forms of IL-2R complex are found on NK cells, memory T cells, regulatory T cells and activated T cells, leading to pleiotrophic effects of IL-2, dependent on concentration of cytokine and dominance of different cell populations and concurrent alternative signals in any given tissue (diseased or normal).
  • Overstimulation of regulatory T cells in cancer therapy with IL-2 may inhibit the desired stimulation of cytotoxic T effector cells.
  • rapid over- stimulation of effector T cells throughout the system may lead to dramatic toxicity.
  • This intrinsic complexity of IL2 signaling on different cell classes has compounded the difficulty of using IL-2 as a soluble protein therapeutic.
  • IL-2 proteins For use as potential therapeutic agents, IL-2 proteins have commonly been synthesized as free cytokine, or antibody fusion proteins, but these molecules have suffered from high toxicity, poor biodistribution and/or poor therapeutic index.
  • IL-2 and IL-2 antibody fusions could be made more tolerable and effective therapeutic agents e.g., by improving their half-life in man, coupled with reduced binding affinity to one or more of its 3 known receptors. This would allow the effective stimulation of the desired effector cells of the immune system, while minimizing stimulation of undesired cells.
  • a molecule which has reduced or eliminated affinity for CD25, coupled with conditional affinity for CD 122 may exhibit 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 a cancer setting where one would seek to stimulate Teff cells over Treg, thereby stimulating anti-cancer therapeutic activity.
  • a molecule which has native (high) affinity for CD25, coupled with reduced affinity for CD 122 may exhibit 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 an inflammatory disease setting where one would seek to stimulate Treg cells over Teff, thereby stimulating anti-inflammatory therapeutic activity.
  • IE-2 derived molecules with the correct combination of expression characteristics, purification quality, stability, solubility, and controlled receptor signaling is far from trivial.
  • An ideal method would combine facile expression of the protein in the highly established CHO cell platform, purification via standard manufacturing column processes and site- specific conjugation at an optimal site (or sites), with a moiety that modulates both receptor binding activity and pharmacokinetics.
  • the Inventors have developed new mutant IL-2, derived from human IL-2, comprising non-endogenous transglutaminase motifs. Said transglutaminase motifs enable to conjugate moieties in a simple and reproducible way, by transglutamination.
  • the present invention relates to a mutant IL-2 derived from human IL-2 with the sequence SEQ ID NO: 1 or from a variant thereof, comprising at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif is inserted by: insertion of a Q residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, El 10 and S130, and/or insertion of a motif at the N-terminus or C-terminus, wherein said motif comprises a sequence selected from the group comprising or consisting of LQS and TQG, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e.,
  • said at least one non-endogenous transglutaminase motif is inserted by insertion of a Q residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, S130.
  • said at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of XiLQ wherein Xi is A, L, H, I or V; NX 2 Q wherein X 2 is Y, H or F; YRQ, LTQ; AQX 3 wherein X 3 is A or E, TEQ, LFQ; GSQ and VIQ , preferably comprising or consisting of XiLQ wherein Xi is L, H, I or V; NX 2 Q wherein X 2 is Y, H or F; YRQ, LTQR (SEQ ID NO: 2); LFQ; GSQ and VIQ.
  • said at least one non-endogenous transglutaminase motif comprises or consists of: a sequence selected from the group comprising or consisting of LEHLLQD (SEQ ID NO: 3), LEHLQSD (SEQ ID NO: 4), LLQLLLD (SEQ ID NO: 5), MILQGIN (SEQ ID NO: 6), MLLQGIN (SEQ ID NO: 7), NNYQNP (SEQ ID NO: 8), NNHQNP (SEQ ID NO: 9), LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), LLQC (SEQ ID NO: 13), KNFQLRPRD (SEQ ID NO: 14), KLFQLRPRD (SEQ ID NO: 15), KLLQLRPRD (SEQ ID NO: 16), VLQ, LLQ, GSQTT (SEQ ID NO: 17), GSQST (SEQ ID NO: 18), V
  • said at least one non-endogenous transglutaminase motif is inserted by: insertion of a Q residue at one or more of the following positions by reference to SEQ ID NO: 1: N33, T41, F42, H79, E100, and /or insertion of a motif at the N-terminus or C-terminus, wherein said motif comprises or consists of a sequence selected from the group comprising or consisting of LQS and TQG.
  • said at least one non-endogenous transglutaminase motif comprises or consists of: a sequence selected from the group comprising or consisting of LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), KLFQLRPRD (SEQ ID NO: 15), KLLQLRPRD (SEQ ID NO: 16), GSQST (SEQ ID NO: 18), NNYRQP (SEQ ID NO: 102) and NRYRQP (SEQ ID NO: 103), and/or a sequence selected from the group comprising or consisting of LQS and TQG, preferably wherein said sequence is added at the C-terminus or at the N-terminus.
  • said at least one non-endogenous transglutaminase motif comprises or consists of: a sequence selected from the group comprising or consisting of LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), KLFQLRPRD (SEQ ID NO: 15), KLLQLRPRD (SEQ ID NO: 16), GSQST (SEQ ID NO: 18), NNYRQP (SEQ ID NO: 102) and NRYRQP (SEQ ID NO: 103) , and/or a sequence selected from the group comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), LQSPTSSSTKKTQ (SEQ ID NO: 42), and TQGASSTKKTQ (SEQ ID NO: 44), preferably wherein said sequence is added at the N-terminus, and/or a sequence selected from the group comprising or consisting of GGLQSP (SEQ ID NO: 10),
  • the mutant comprises or consists of a sequence selected from the group comprising or consisting of 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 comprising or consisting of SEQ ID NO: 19 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63 and SEQ ID NOs: 110 and 111.
  • the mutant comprises or consists of a sequence selected from the group comprising or consisting of 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 comprising or consisting of SEQ ID NO: 19 to SEQ ID NO: 39, SEQ ID NO: 51 to SEQ ID NO: 63 and SEQ ID NOs: 110 and 111.
  • the mutant comprises or consists of a sequence selected from the group comprising or consisting of 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: SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 58 to SEQ ID NO: 63 and SEQ ID NOs: 110 and 111.
  • the mutant comprises or consists of a sequence selected from the group comprising or 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 NOs: 110 and 111.
  • the mutant IL-2 further comprises a substitution of the F42 residue by reference to SEQ ID NO: 1 into any other amino acid, more preferably a substitution of the F42 residue into an alanine residue.
  • the present invention also relates to a fusion protein comprising a mutant IL-2 as described hereinabove, and further comprising at least one masking moiety, wherein the at least one masking moiety is linked to a Q residue of the at least one non-endogenous transglutaminase motif of the mutant IL-2 with a cleavable linker.
  • the fusion protein further comprises an antibody or antigenbinding fragment thereof or an antibody mimetic, preferably wherein the antibody or antigen-binding fragment thereof or antibody mimetic is linked to the mutant IL-2 with a linker.
  • Antibody and “immunoglobulin”, as used herein, may be used interchangeably and refer to a protein having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. “Antibodies” refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest. Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood. The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. Although the following discussion will generally be directed to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention.
  • immunoglobulins comprise two identical light polypeptide chains of molecular weight of about 23 kDa, and two identical heavy chains of molecular weight of about 53-70 kDa.
  • the four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
  • the light chains of an antibody are classified as either kappa (K) or lambda ( ).
  • K kappa
  • Each heavy chain class may be bonded with either a K or light chain.
  • the light and heavy chains are covalently bonded to each other, and the “tail” regions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • heavy chains are classified as gamma (y), mu (p), alpha (a), delta (8) or epsilon (a) with some subclasses among them (e.g., yl- y4).
  • immunoglobulin subclasses or “isotypes” e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc.
  • isotypes e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc.
  • Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the present invention.
  • the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens.
  • VL domain light chain variable domain
  • VH domain heavy chain variable domain
  • This quaternary antibody structure forms the antigen binding site presents 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 of the VH and VL chains.
  • CDRs complementarity determining regions
  • Antigen-binding fragment refers to a part or region of an antibody which comprises fewer amino acid residues than the whole antibody.
  • An “antigen-binding fragment” binds antigen and/or competes with the whole antibody from which it derives for antigen binding (e.g., specific binding to human CD25).
  • Antigenbinding fragments encompasses, without any limitation, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a nanobody, a domain antibody, and a unibody.
  • “Derived from” indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of mutant IL-2 that is derived from human IL-2, the mutant retains sufficient IL-2 structure such that it has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connote or include a limitation to a particular process of producing the mutant IL-2.
  • Diabodies refer to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL such that inter-chain but not intra-chain pairing of the variable domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL of the two antibodies are present on different polypeptide chains.
  • Diabodies are described, for example, in patent EP0404097 or patent application WO 1993011161.
  • Domain antibodies refer to the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies.
  • Fab refers to a monovalent fragment containing the following regions: VH, VL, CHI and CL, linked by an intramolecular disulfide bond.
  • F(ab')2 refers to a fragment containing two antigen-binding regions joined by disulfides bonds.
  • Fab refers to a fragment obtained by the reduction of F(ab')2 fragments.
  • Fd fragment refers to the heavy chain of the Fab fragment, comprising the VH and CHI regions.
  • Fv refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one VH and one VL in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the heavy and light chain) that contribute to antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Humanized antibody or antigen-binding fragment thereof refers to a chimeric antibody or antigen-binding fragment thereof which contains minimal sequence derived from a non-human immunoglobulin. It includes antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell, e.g. , by altering the non- human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • Humanized antibodies or antigen-binding fragments thereof comprised in a fusion protein according to the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • humanized antibody or antigenbinding fragment thereof also includes antibodies and antigen-binding fragment thereof in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • humanized antibody or antigen-binding fragment thereof may refer to an antibody or antigen-binding fragment thereof in which the CDRs of a recipient human antibody are replaced by CDRs from a donor non-human antibody.
  • Humanized antibodies or antigen-binding fragments thereof may also comprise residues of donor origin in the framework sequences.
  • the humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of a human immunoglobulin constant region.
  • Humanized antibodies or antigen-binding fragments thereof may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed using methods known in the art (e.g., Jones et al., 1986. Nature.
  • Identity when used herein in a relationship between the sequences of two or more amino acid sequences, or of two or more nucleic acid sequences, refers to the degree of sequence relatedness between 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. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988).
  • Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt l):387-95), BLASTP, BLASTN, and FASTA (Altschul et al., 1990. J Mol Biol. 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 may also be used to determine identity.
  • GAP Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux et al., 1984. Nucleic Acids Res. 12(1 Pt
  • Interleukin-2 refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated.
  • the term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell.
  • the term also encompasses naturally occurring variants of IL-2 (e.g., splice variants or allelic variants) as well as truncated forms of IL-2.
  • IL-2 is human IL-2, having the sequence SEQ ID NO: 1.
  • IL-2 is a truncated human IL-2, having the sequence SEQ ID NO: 50.
  • Interleukin-2 receptor refers to the heterotrimeric receptor of IL- 2. This receptor is composed by different combinations of the following chains: a (alpha) chain (also called IL-2Ra, CD25, or Tac antigen), P (beta) chain (also called IL-2RP, or CD122), and y (gamma) chain (also called IL-2Ry, common gamma chain, or CD132). Depending on the combinations of these chains, IL-2 receptors having low, intermediate, or high affinity for IL-2 may be generated. Low-affinity IL-2 receptors are composed of a IL-2Ra chain. Intermediate- affinity IL-2 receptors are composed of IL-2RP and IL-2Ry chains. High-affinity IL-2 receptors are composed of IL-2Ra, IL-2RP and IL-2Ry chains.
  • a (alpha) chain also called IL-2Ra, CD25, or Tac antigen
  • P (beta) chain also called IL-2RP, or CD122
  • IL-2 mutant or “mutant IL-2” refers to an IL-2 that displays modifications in sequence and/or in functional properties.
  • “Mammal” refers to any mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is a human.
  • Peptide refers to a linear polymer of amino acids of at least 2 amino acids and less than 50 amino acids linked together by peptide bonds.
  • Polyethylene glycol or ‘ ‘PEG” refers to a biocompatible, synthetic, hydrophilic polyether compound with the formula hereinabove, wherein n > 4.
  • PEG polyethylene oxide
  • POE polyoxyethylene
  • Single chain antibody refers to any antibody or fragment thereof that is a protein having a primary structure comprising or consisting of one uninterrupted sequence of contiguous amino acid residues, including without limitation (1) single-chain Fv molecules (scFv); (2) single chain proteins containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety; and (3) single chain proteins containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety.
  • scFv single chain proteins containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety
  • Single-chain Fv also abbreviated as “sFv” or “scFv”, refers to antibody fragments that comprise the VH and VL antibody domains connected into a single amino acid chain.
  • the scFv amino acid sequence further comprises a peptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • Small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • Subject refers to a mammal, preferably a human.
  • a subject may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.
  • substantially human in the context of the constant region of a humanized or chimeric antibody or antigen-binding fragment thereof, refers to an amino acid sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more with a human constant region.
  • human amino acid sequence in this context, refers to an amino acid sequence which is encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes.
  • the present invention also contemplates proteins comprising constant domains of “human” sequence which have been altered, by one or more amino acid additions, deletions or substitutions with respect to the human sequence, excepting those embodiments where the presence of a “fully human hinge region” is expressly required.
  • the presence of a “fully human hinge region” in the at least one antibody or antigenbinding fragment thereof comprised in a fusion protein according to the present invention may be beneficial both to minimize immunogenicity and to optimize stability of the antibody. It is considered that one or more amino acid substitutions, insertions or deletions may be made within the constant region of the heavy and/or the light chain, particularly within the Fc region.
  • Amino acid substitutions may result in replacement of the substituted amino acid with a different naturally occurring amino acid, or with a nonnatural or modified amino acid.
  • Other structural modifications are also permitted, such as for example changes in glycosylation pattern (e.g., by addition or deletion of N- or O- linked glycosylation sites).
  • “Therapeutically effective amount” refers to the level or amount of an antibody as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition.
  • a therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.
  • a subject is successfully "treated” for a cancer if, after receiving a therapeutic amount of at least one mutant IL-2, fusion protein, nucleic acid or expression vector according to the present invention, the subject shows at least one of the following: reduction in the number of cancer cells (or tumor size) or pathogenic cells; reduction in the percent of total cells that are cancerous or pathogenic; relief to some extent of one or more of the symptoms associated with the cancer or the infectious disease to be treated; reduced morbidity and mortality; and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • Unibodies refer to an antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies.
  • Variant refers to nucleic acid or amino acid sequences that typically differs from a nucleic acid or an amino acid sequence specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions.
  • percentage of identity it is meant a nucleic acid or amino acid sequence comprising at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference nucleic acid or amino acid sequence.
  • This invention relates to mutants of IL-2 comprising at least one transglutaminase site (TG) enabling to conjugate moieties in a simple and reproducible way by transglutamination.
  • TG transglutaminase site
  • this concept of adding TG sites to conjugate moieties may be equally applied to other molecules, such as, for example, IL-2 mimetics.
  • other types of conjugation via nonnatural amino acids and the introduction of other types of coupling sites may be applied at the sites identified within.
  • the mutant IL-2 of the invention may exhibit reduced or eliminated affinity for CD25, coupled with modulated affinity for CD 122, in which the modulated affinity for CD 122 is achieved via transglutaminase-mediated conjugation at one or more single site(s) newly introduced to the IL-2 structure via mutagenesis.
  • the mutant IL-2 may be conjugated to a moiety, such as PEG via a linker which is, or is not, selectively cleaved in diseased tissue. Without willing to be bound to any theory, it is hypothesized that this form of mutant IL-2 may exhibit reduced ability to stimulate T regulatory (Treg) cells (which are CD25-high) while effectively stimulate T effector (Teff) cells (CD25-low). This is a potentially beneficial phenotype in a cancer setting where one would seek to stimulate Teff cells over Treg, thereby stimulating anti-cancer therapeutic activity.
  • T regulatory T regulatory
  • Teff T effector
  • the mutant IL-2 of the invention may exhibit native (high) affinity for CD25, coupled with modulated affinity for CD 122, in which the modulated affinity for CD 122 is achieved via transglutaminase-mediated conjugation at one mor more single site(s) newly introduced to the IL-2 structure via mutagenesis.
  • the mutant IL-2 may be conjugated to a moiety, such as PEG via a linker which is, or is not, selectively cleaved in diseased tissue.
  • a linker which is, or is not, selectively cleaved in diseased tissue.
  • it is hypothesized that the mutant IL-2 may exhibit enhanced ability to stimulate Treg cells (which are CD25-high) and minimized stimulation of Teff cells (CD25-low). This is a potentially beneficial phenotype in an inflammatory disease setting where one would seek to stimulate Treg cells over Teff, thereby stimulating anti-inflammatory therapeutic activity.
  • the present invention relates to a mutant IL-2, preferably a mutant IL-2 derived from human IL-2.
  • human IL-2 comprises or consists of the sequence with SEQ ID NO: 1 or a variant thereof, preferably wherein said variant comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1.
  • the mutant IL-2 of the present invention is derived from human IL-2 with SEQ ID NO: 1 or a variant thereof, preferably wherein said variant comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1.
  • Said variant may comprise the same number of amino acids as SEQ ID NO: 1, and thus the mutations and positions described herein are the same for the variant.
  • said variant may comprise a different number of amino acids as SEQ ID NO: 1.
  • the skilled artisan in the art will know how to place the mutations and positions described herein in the variant.
  • the mutant IL-2 comprises a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e. F42A mutation), by reference to SEQ ID NO: 1.
  • the IL-2 mutant has a reduced binding or affinity for the IL- 2R alpha chain as compared to the IL-2 from which it derives.
  • the IL-2 mutant comprises one or more of the followings modifications: a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation), by reference to SEQ ID NO: 1, and/or a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation), by reference to SEQ ID NO: 1, and/or a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e. F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif and a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e. F42A mutation), by reference to SEQ ID NO: 1.
  • the present invention also relates to a mutant IL-2, preferably derived from the human IL-2 with SEQ ID NO: 1, or a variant thereof, comprising at least one non-endogenous transglutaminase motif, and further comprising a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e. F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises one non-endogenous transglutaminase motif. In one embodiment, the mutant IL-2 comprises two or more non- endogenous transglutaminase motifs.
  • Transglutaminase motifs may be found endogenously in proteins, and in particular, in IL-2.
  • human IL-2 with SEQ ID NO: 1 comprises an endogenous transglutaminase motif with the Q74 residue, and with the motif consisting of AQS.
  • a non-endogenous transglutaminase motif is a transglutaminase motif that is not found in the IL-2 from which the IL-2 mutant is derived.
  • the mutant IL-2 derived from the human IL-2 with SEQ ID NO: 1 or a variant thereof does not comprise non-endogenous transglutaminase motifs that are different from the ones described herein.
  • the mutant IL-2 derived from the human IL-2 with SEQ ID NO: 1 or a variant thereof comprises one or more non-endogenous transglutaminase motif(s) as described herein and does not comprise other non- endogenous transglutaminase motifs.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif inserted within the IL-2 sequence.
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, El 10 and S130.
  • Q glutamate
  • the at least one non-endogenous transglutaminase motif is characterized by the presence of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, El 10 and S130.
  • Q glutamate
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, and S 130.
  • Q glutamate
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: N33, T41, F42, H79 and E100.
  • the at least one non-endogenous transglutaminase motif is inserted by substitution of one or more of the following amino acids by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, H79, E95, E100, E110 and S130, with a Q residue.
  • the at least one non-endogenous transglutaminase motif is inserted by substitution of one or more of the following amino acids by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, H79, E95, E100, and S130, with a Q residue.
  • the at least one non-endogenous transglutaminase motif is inserted by modifying the amino acids surrounding the Q57 residue by reference to SEQ ID NO: 1, in particular, one or more amino acid(s) surrounding the Q57 residue.
  • the amino acids surroundings the Q57 residue are mutated, preferably substituted by other amino acids, to form a transglutaminase motif.
  • the at least one non-endogenous transglutaminase motif is inserted by: substitution of a Q residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, H79, E95, E100, and S130, and/or substitution of one or more amino acid(s) surrounding the Q57 residue by reference to SEQ ID NO: 1 to form a transglutaminase motif.
  • the at least one non-endogenous transglutaminase motif is inserted by substitution of the amino acid H55 by reference to SEQ ID NO: 1 with a leucine residue (z.e. H55L mutation), for the Q57 position.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of XiLQ wherein Xi is A, L, H, I or V; NX 2 Q wherein X 2 is Y, H or F; YRQ, LTQ; AQX 3 wherein X 3 is A or E, TEQ, LFQ; GSQ and VIQ.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of XiLQ wherein Xi is L, H, I or V; NX 2 Q wherein X 2 is Y, H or F; YRQ, LTQR (SEQ ID NO: 2); LFQ; GSQ and VIQ.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LLQ, YRQ, LTQR (SEQ ID NO: 2), LFQ and GSQ.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LEHLLQD (SEQ ID NO: 3), LEHLQSD (SEQ ID NO: 4), LLQLLLD (SEQ ID NO: 5), MILQGIN (SEQ ID NO: 6), MLLQGIN (SEQ ID NO: 7), NNYQNP (SEQ ID NO: 8), NNHQNP (SEQ ID NO: 9), LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), LLQC (SEQ ID NO: 13), KNFQLRPRD (SEQ ID NO: 14), KLFQLRPRD (SEQ ID NO: 15), KLLQLRPRD (SEQ ID NO: 16), VLQ, LLQ, GSQTT (SEQ ID NO: 17), GSQST (SEQ ID NO: 18), VIQ
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LEHLLQD (SEQ ID NO: 3), LEHLQSD (SEQ ID NO: 4), LLQLLLD (SEQ ID NO: 5), MILQGIN (SEQ ID NO: 6), MLLQGIN (SEQ ID NO: 7), NNYQNP (SEQ ID NO: 8), NNHQNP (SEQ ID NO: 9), LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), LLQC (SEQ ID NO: 13), KNFQLRPRD (SEQ ID NO: 14), KLFQLRPRD (SEQ ID NO: 15), KLLQLRPRD (SEQ ID NO: 16), VLQ, LLQ, GSQTT (SEQ ID NO: 17), GSQST (SEQ ID NO: 18), VIQ
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, El 10 and S130, and comprises or consists of a sequence selected from the group comprising or consisting of XiLQ wherein Xi is A, L, H, I or V; NX2Q wherein X 2 is Y, H or F; YRQ, LTQ; AQX 3 wherein X 3 is A or E; TEQ; LFQ; GSQ and VIQ.
  • Q glutamate
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, El 10 and S130, and comprises or consists of a sequence selected from the group comprising or consisting of LEHLLQD (SEQ ID NO: 3), LEHLQSD (SEQ ID NO: 4), LLQLLLD (SEQ ID NO: 5), MILQGIN (SEQ ID NO: 6), MLLQGIN (SEQ ID NO: 7), NNYQNP (SEQ ID NO: 8), NNHQNP (SEQ ID NO: 9), LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), LLQC (Q) residue at one or more
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, and S130, and comprises or consists of a sequence selected from the group comprising or consisting of XiLQ wherein Xi is L, H, I or V; NX2Q wherein X2 is Y, H or F; YRQ; LTQR (SEQ ID NO: 2); LFQ; GSQ and VIQ.
  • Q glutamate
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, Q57, H79, E95, E100, and S130, and comprises or consists of a sequence selected from the group comprising or consisting of LEHLLQD (SEQ ID NO: 3), LEHLQSD (SEQ ID NO: 4), LLQLLLD (SEQ ID NO: 5), MILQGIN (SEQ ID NO: 6), MLLQGIN (SEQ ID NO: 7), NNYQNP (SEQ ID NO: 8), NNHQNP (SEQ ID NO: 9), LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), LLQC (SEQ ID NO: 13), K
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: N33, T41, F42, H79 and E100, and comprises or consists of a sequence selected from the group comprising or consisting of LLQ, YRQ, LTQR (SEQ ID NO: 2), LFQ and GSQ.
  • Q glutamate
  • the at least one non-endogenous transglutaminase motif is inserted by insertion of a glutamate (Q) residue at one or more of the following positions by reference to SEQ ID NO: 1: N33, T41, F42, H79 and E100, and comprises or consists of a sequence selected from the group comprising or consisting of LLQFKF (SEQ ID NO: 10), LLQAKF (SEQ ID NO: 11), MLTQRF (SEQ ID NO: 12), KLFQLRPRD (SEQ ID NO: 15), KLLQLRPRD (SEQ ID NO: 16), GSQST (SEQ ID NO: 18), NNYRQP (SEQ ID NO: 102) and NRYRQP (SEQ ID NO: 103).
  • Q glutamate
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the L19 position by reference to SEQ ID NO: 1 (z.e.
  • the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., a C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the L18 position by reference to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the L18 position by reference to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the L18 position by reference to SEQ ID NO:
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (z.e.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 20.
  • An example of such mutant IL-2 is the mutant 2-203.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 156.
  • An example of such mutant IL-2 is the mutant 2-597.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 131.
  • An example of such mutant IL-2 is the mutant 2-573.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 21.
  • An example of such mutant IL-2 is the mutant 2-204.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 157.
  • An example of such mutant IL-2 is the mutant 2-598.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 132.
  • An example of such mutant IL-2 is the mutant 2-574.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the N26 position by reference to SEQ ID NO: 1 (i.e.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 23.
  • An example of such mutant IL-2 is the mutant 2-206.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 159.
  • An example of such mutant IL-2 is the mutant 2-600.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 134.
  • An example of such mutant IL-2 is the mutant 2-576.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the K32 position by reference to SEQ ID NO: 1 (z.e. comprises a Q32 residue, preferably obtained by a K32Q mutation), and comprises or consists of the sequence NYQ, preferably comprises or consists of the sequence NNYQNP (SEQ ID NO: 8), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 24.
  • An example of such mutant IL-2 is the mutant 2-207.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 160.
  • An example of such mutant IL-2 is the mutant 2-601.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 135.
  • An example of such mutant IL-2 is the mutant 2-577.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the K32 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 25.
  • An example of such mutant IL-2 is the mutant 2-208.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 161.
  • An example of such mutant IL-2 is the mutant 2-602.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 136.
  • An example of such mutant IL-2 is the mutant 2-578.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the N33 position by reference to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the N33 position by reference to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the N33 position by reference to SEQ ID NO:
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e. C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (z.e.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 110.
  • An example of such mutant IL-2 is the mutant 2-103.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 162.
  • An example of such mutant IL-2 is the mutant 2-603.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 137.
  • An example of such mutant IL-2 is the mutant 2-579.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the N33 position by reference to SEQ ID NO: 1 (z.e. comprises a Q33 residue, preferably obtained by a N33Q mutation), and comprises or consists of the sequence YRQ, preferably comprises or consists of the sequence NRYRQP (SEQ ID NO: 103), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e.
  • C125S mutation (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (z.e. Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 111.
  • An example of such mutant IL-2 is the mutant 2-104.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 163.
  • An example of such mutant IL-2 is the mutant 2-604.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 138.
  • An example of such mutant IL-2 is the mutant 2-580.
  • the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the F42 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 28.
  • An example of such mutant IL-2 is the mutant 2-107.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the F42 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 112.
  • An example of such mutant IL-2 is the mutant 2-108.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 165.
  • An example of such mutant IL-2 is the mutant 2-606.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the T51 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 113.
  • An example of such mutant IL-2 is the mutant 2-109.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 166.
  • An example of such mutant IL-2 is the mutant 2-607.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 140.
  • An example of such mutant IL-2 is the mutant 2-582.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the L53 position by reference to SEQ ID NO: 1 (i.e.
  • the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 168.
  • An example of such mutant IL-2 is the mutant 2-609.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 142.
  • An example of such mutant IL-2 is the mutant 2-584.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the Q57 position by reference to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the Q57 position by reference to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the Q57 position by reference to SEQ ID NO:
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 29.
  • An example of such mutant IL-2 is the mutant 2-209.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 169.
  • An example of such mutant IL-2 is the mutant 2-610.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 143.
  • An example of such mutant IL-2 is the mutant 2-585.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (z.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 30.
  • An example of such mutant IL-2 is the mutant 2-210.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 170.
  • An example of such mutant IL-2 is the mutant 2-611.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 144.
  • An example of such mutant IL-2 is the mutant 2-222.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (i.e.
  • the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the mutant IL-2
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 32.
  • An example of such mutant IL-2 is the mutant 2-114.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue i.e., F42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., a C125S mutation) and a substitution of the glutamine
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 64.
  • An example of such mutant IL-2 is the mutant 2-132.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (i.e., comprises a Q79 residue, preferably obtained by a H79Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence KLLQLRPRD (SEQ ID NO: 16), and wherein the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 65.
  • An example of such mutant IL-2 is the mutant 2-133.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the E95 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 35.
  • An example of such mutant IL-2 is the mutant 2-211.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 171.
  • An example of such mutant IL-2 is the mutant 2-612.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 145.
  • An example of such mutant IL-2 is the mutant 2-586.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the E95 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a Q residue at the E100 position by reference to SEQ ID NO: 1 (z.e.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 173.
  • An example of such mutant IL-2 is the mutant 2-614.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 66.
  • An example of such mutant IL-2 is the mutant 2-134.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 117.
  • An example of such mutant IL-2 is the mutant 2-118.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 176.
  • An example of such mutant IL-2 is the mutant 2-617.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 177.
  • An example of such mutant IL-2 is the mutant 2-618.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 150.
  • An example of such mutant IL-2 is the mutant 2-591.
  • the mutant IL-2 of the present invention preferably derived from human IL-2 with SEQ ID NO: 1 or a variant thereof, comprises at least one non- endogenous transglutaminase motif that is inserted at the N-terminus or C-terminus of the IL-2 sequence.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LQS and TQG, preferably wherein the sequence is present or added at the N- or C- terminus.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), TQGAPTSSSTKKTQ (SEQ ID NO: 41), LQSPTSSSTKKTQ (SEQ ID NO: 42), TQGASSSTKKTQ (SEQ ID NO: 43), TQGASSTKKTQ (SEQ ID NO: 44), TQGASTKKTQ (SEQ ID NO: 45), GGTQGA (SEQ ID NO: 46), GGLQSP (SEQ ID NO: 47), GLQSP (SEQ ID NO: 48), LQSP (SEQ ID NO: 49), and QGA, preferably wherein said sequence is present or added at the N- or C-terminus.
  • LQSPGAPTSSSTKKTQ SEQ ID NO: 40
  • TQGAPTSSSTKKTQ SEQ ID NO: 41
  • LQSPTSSSTKKTQ SEQ ID NO: 42
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), TQGAPTSSSTKKTQ (SEQ ID NO: 41), LQSPTSSSTKKTQ (SEQ ID NO: 42), TQGASSSTKKTQ (SEQ ID NO: 43), TQGASSTKKTQ (SEQ ID NO: 44), and TQGASTKKTQ (SEQ ID NO: 45), preferably wherein said sequence is present or added at the N-terminus.
  • LQSPGAPTSSSTKKTQ SEQ ID NO: 40
  • TQGAPTSSSTKKTQ SEQ ID NO: 41
  • LQSPTSSSTKKTQ SEQ ID NO: 42
  • TQGASSSTKKTQ SEQ ID NO: 43
  • TQGASSTKKTQ SEQ ID NO: 44
  • TQGASTKKTQ SEQ ID NO: 45
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), LQSPTSSSTKKTQ (SEQ ID NO: 42), and TQGASSTKKTQ (SEQ ID NO: 44), preferably wherein said sequence is present or added at the N-terminus.
  • the at least one non-endogenous transglutaminase motif defined hereinabove is inserted at the N-terminus of an IL-2 sequence with SEQ ID NO: 50.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or 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 said sequence is present or added at the C-terminus.
  • the at least one non-endogenous transglutaminase motif comprises or consists of a sequence selected from the group comprising or consisting of GGLQSP (SEQ ID NO: 47), GLQSP (SEQ ID NO: 48), LQSP (SEQ ID NO: 49) and QGA, preferably wherein said sequence is present or added at the C-terminus.
  • the at least one non-endogenous transglutaminase motif defined hereinabove is inserted at the C-terminus of the IL-2 sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), is inserted at the N-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 51.
  • An example of such mutant IL-2 is the mutant 2-120.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 178.
  • An example of such mutant IL-2 is the mutant 2-619.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), at the N-terminus), and wherein the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue i.e., F42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 67.
  • An example of such mutant IL-2 is the mutant 2-135.
  • the mutant IE-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGAPTSSSTKKTQ (SEQ ID NO: 41), at the N-terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGAPTSSSTKKTQ (SEQ ID NO: 41), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGAPTSSSTKKTQ (SEQ ID NO: 41), is inserted at the N-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGAPTSSSTKKTQ (SEQ ID NO: 41), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 thereof comprises or consists of the sequence with SEQ ID NO: 52.
  • An example of such mutant IL-2 is the mutant 2-215.
  • the mutant IL-2 thereof comprises or consists of the sequence with SEQ ID NO: 179.
  • An example of such mutant IL-2 is the mutant 2-620.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 151.
  • An example of such mutant IL-2 is the mutant 2-592.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 53.
  • An example of such mutant IL-2 is the mutant 2-121.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 180.
  • An example of such mutant IL-2 is the mutant 2-621.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus (z.e., comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus), and wherein the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alan
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 68.
  • An example of such mutant IL-2 is the mutant 2-136.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSSTKKTQ (SEQ ID NO: 43), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSSTKKTQ (SEQ ID NO: 43), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (z.e.
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGASSSTKKTQ (SEQ ID NO: 43), is inserted at the N-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGASSSTKKTQ (SEQ ID NO: 43), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 54.
  • An example of such mutant IL-2 is the mutant 2-216.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 181.
  • An example of such mutant IL-2 is the mutant 2-622.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 152.
  • An example of such mutant IL-2 is the mutant 2-593.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the sequence comprising or consisting of TQG is inserted at the N-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of TQG is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 55.
  • An example of such mutant IL-2 is the mutant 2-122.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 182.
  • An example of such mutant IL-2 is the mutant 2-623.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus (i.e.
  • mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a gluta glutamine 74 residue with any other amino acid residue, preferably a gluta glutamine 74 residue with any other amino acid residue, preferably a gluta glutamine 74 residue, preferably a glut
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 69.
  • An example of such mutant IL-2 is the mutant 2-137.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASTKKTQ (SEQ ID NO: 45), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASTKKTQ (SEQ ID NO: 45), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e. C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e.
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGASTKKTQ (SEQ ID NO: 45), is inserted at the N-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of TQG is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • An example of such mutant IL- 2 is the mutant 2-217.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 56.
  • An example of such mutant IL-2 is the mutant 2-217.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 183.
  • An example of such mutant IL-2 is the mutant 2-624.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 153.
  • An example of such mutant IL-2 is the mutant 2-594.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting GGTQGA (SEQ ID NO: 46), at the C- terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of GGTQGA (SEQ ID NO: 46), at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), and/or a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • sequence comprising or consisting of TQG preferably the sequence comprising or consisting of GGTQGA (SEQ ID NO: 46), is inserted at the C-terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 57.
  • An example of such mutant IL-2 is the mutant 2-218.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 184.
  • An example of such mutant IL-2 is the mutant 2-625.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 185.
  • An example of such mutant IL-2 is the mutant 2-626.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), at the C- terminus (z.e., comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), at the C- terminus), and wherein the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., E42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125
  • the mutant IL- comprises or consists of the sequence with SEQ ID NO: 70.
  • An example of such mutant IL-2 is the mutant 2-138.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GLQSP (SEQ ID NO: 48) at the C- terminus (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GLQSP (SEQ ID NO: 48), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GLQSP (SEQ ID NO: 48), is inserted at the C- terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 59.
  • An example of such mutant IL-2 is the mutant 2-124.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 186.
  • An example of such mutant IL-2 is the mutant 2-627.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSP (SEQ ID NO: 49), at the C-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting LQSP (SEQ ID NO: 49), at the C-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSP (SEQ ID NO: 49), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSP (SEQ ID NO: 49), is inserted at the C- terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 60.
  • An example of such mutant IL-2 is the mutant 2-125.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 187.
  • An example of such mutant IL-2 is the mutant 2-628.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSP (SEQ ID NO: 49), at the C-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSP (SEQ ID NO: 49), at the C-terminus), and wherein the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue i.e., F42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 71.
  • An example of such mutant IL-2 is the mutant 2-140.
  • the mutant IL-2 comprises at least one non-endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of QGA, at the C- terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of QGA at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., Q74E mutation) or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of QGA is inserted at the C-terminus of the sequence with SEQ ID NO: 1.
  • the sequence comprising or consisting of QGA is inserted at the C-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 61.
  • An example of such mutant IL-2 is the mutant 2-126.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 188.
  • An example of such mutant IL-2 is the mutant 2-629.
  • the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 comprises at least one non- endogenous transglutaminase motif as compared to SEQ ID NO: 1 or variant thereof, wherein said motif: is inserted by insertion of a sequence comprising or consisting of QGA, at the C- terminus (i.e., comprises a sequence comprising or consisting of QGA at the C- terminus), and wherein the mutant IL-2 comprises a substitution of the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue i.e., F42A mutation), by reference to SEQ ID NO: 1, optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cyste
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 72.
  • An example of such mutant IL-2 is the mutant 2-141.
  • the present invention further relates to a mutant IL-2 derived from the human IL-2 with SEQ ID NO: 1 or a variant thereof, comprising at least two non-endogenous transglutaminase motifs.
  • the mutant IL-2 derived from the human IL-2 with SEQ ID NO: 1 or a variant thereof further comprises a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e. C125S mutation), by reference to SEQ ID NO: 1.
  • the mutant IL-2 derived from the human IL-2 with SEQ ID NO: 1 or a variant thereof further comprises a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e. Q74E mutation), by reference to SEQ ID NO: 1.
  • the at least two non-endogenous transglutaminase motifs are as defined hereinabove.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e. C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus), optionally wherein the mutant IL-2 comprises a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • LTQR preferably comprises or consists of the sequence MLTQRF (SEQ ID NO: 12), and wherein another motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the F42 position by reference to SEQ ID NO: 1 (i.e.
  • LTQR preferably comprises or consists of the sequence MLTQRF (SEQ ID NO: 12), and wherein another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), at the C- terminus (i.e.
  • sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), is inserted at the C-terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the F42 position by reference to SEQ ID NO: 1 (i.e.
  • LTQR preferably comprises or consists of the sequence MLTQRF (SEQ ID NO: 12)
  • MLTQRF SEQ ID NO: 12
  • another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GLQSP (SEQ ID NO: 48) at the C- terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GLQSP (SEQ ID NO: 48), at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GLQSP (SEQ ID NO: 48), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 88.
  • An example of such mutant IL-2 is the mutant 2-159.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the E42 position by reference to SEQ ID NO: 1 (i.e.
  • LTQR preferably comprises or consists of the sequence MLTQRF (SEQ ID NO: 12)
  • MLTQRF SEQ ID NO: 12
  • another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSP (SEQ ID NO: 49), at the C-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting LQSP (SEQ ID NO: 49), at the C-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSP (SEQ ID NO: 49), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 89.
  • An example of such mutant IL-2 is the mutant 2-160.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the E42 position by reference to SEQ ID NO: 1 (i.e. comprises a Q42 residue, preferably obtained by a F42Q mutation), and comprises or consists of the sequence LTQR (SEQ ID NO: 2), preferably comprises or consists of the sequence MLTQRF (SEQ ID NO: 12), and wherein another motif: is inserted by insertion of a sequence comprising or consisting of QGA, at the C- terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of QGA at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of QGA is inserted at the C-terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 90.
  • An example of such mutant IL-2 is the mutant 2-161.
  • the mutant IL-2 comprises at least three non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (i.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQAKF (SEQ ID NO: 11), and wherein another motif: is inserted by insertion of a Q residue at the T51 position by reference to SEQ ID NO: 1 (z.e.
  • a Q51 residue preferably obtained by a T51Q mutation
  • comprises or consists of the sequence AQA preferably comprises or consists of the sequence LAQALK (SEQ ID NO: 106), and wherein another motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (z.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 119.
  • An example of such mutant IL-2 is the mutant 2-127.
  • the mutant IL-2 comprises at least three non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (i.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQAKF (SEQ ID NO: 11), and wherein another motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (i.e.
  • a Q79 residue preferably obtained by a H79Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence KLLQLRPRD (SEQ ID NO: 16)
  • another motif: is inserted by insertion of a Q residue at the El 10 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 120.
  • An example of such mutant IL-2 is the mutant 2-128.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQAKF (SEQ ID NO: 11), and wherein another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), at the C- terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 123.
  • An example of such mutant IL-2 is the mutant 2-148.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (i.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQAKF (SEQ ID NO: 11), and wherein another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GLQSP (SEQ ID NO: 48) at the C- terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GLQSP (SEQ ID NO: 48), at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GLQSP (SEQ ID NO: 48), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 124.
  • An example of such mutant IL-2 is the mutant 2-149.
  • the mutant IL-2 derived from the human IL-2 with SEQ ID NO: 1 or a variant thereof comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10).
  • the other non-endogenous transglutaminase motif is as defined hereinabove.
  • the mutant IE-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10), and wherein another motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (z.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 91.
  • An example of such mutant IL-2 is the mutant 2-162.
  • SEQ ID NO: 91 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRLLQFKFYMPKKATELK HLQCLEEELKPLEEVLNLAESKLFQLRPRDLISNINVIVLELKGSETTFMCEYAD ETATIVEFLNRWITFSQSIISTLT
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10), and wherein another motif: is inserted by insertion of a Q residue at the H79 position by reference to SEQ ID NO: 1 (z.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 92.
  • An example of such mutant IL-2 is the mutant 2-163.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10), and wherein another motif: is inserted by insertion of a Q residue at the E100 position by reference to SEQ ID NO: 1 (z.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 93.
  • An example of such mutant IL-2 is the mutant 2-164.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • a Q41 residue preferably obtained by a T41Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10)
  • another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPTSSSTKKTQ (SEQ ID NO: 42), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 94.
  • An example of such mutant IL-2 is the mutant 2-166.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • a Q41 residue preferably obtained by a T41Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10)
  • another motif: is inserted by insertion of a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of TQG, preferably a sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of TQG preferably the sequence comprising or consisting of TQGASSTKKTQ (SEQ ID NO: 44), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 95.
  • An example of such mutant IL-2 is the mutant 2-167.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • a Q41 residue preferably obtained by a T41Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10)
  • another motif: is inserted by insertion of a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSP (SEQ ID NO: 49), at the C-terminus (i.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting LQSP (SEQ ID NO: 49), at the C-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSP (SEQ ID NO: 49), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 96.
  • An example of such mutant IL-2 is the mutant 2-170.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • a Q41 residue preferably obtained by a T41Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10)
  • another motif: is inserted by insertion of a sequence comprising or consisting of QGA, at the C- terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of QGA at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • sequence comprising or consisting of QGA is inserted at the C-terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 97.
  • An example of such mutant IL-2 is the mutant 2-171.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e. comprises a Q41 residue, preferably obtained by a T41Q mutation), and comprises or consists of the sequence LLQ, preferably comprises or consists of the sequence LLQFKF (SEQ ID NO: 10), and wherein another motif: is inserted by insertion of a Q residue at the T51 position by reference to SEQ ID NO: 1 (i.e.
  • mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (i.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 121.
  • An example of such mutant IL-2 is the mutant 2-129.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • a Q41 residue preferably obtained by a T41Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence preferably LLQFKF (SEQ ID NO: 10)
  • another motif: is inserted by insertion of a sequence comprising or consisting of EQS, preferably a sequence comprising or consisting of the sequence LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), at the N-terminus (z.e.
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), at the N-terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of LQSPGAPTSSSTKKTQ (SEQ ID NO: 40), is inserted at the N-terminus of the sequence with SEQ ID NO: 50.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 125.
  • An example of such mutant IL-2 is the mutant 2-165.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • a Q41 residue preferably obtained by a T41Q mutation
  • comprises or consists of the sequence LLQ preferably comprises or consists of the sequence preferably LLQFKF (SEQ ID NO: 10)
  • another motif: is inserted by insertion of a sequence comprising or consisting of EQS, preferably a sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), at the C- terminus (z.e.
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GGLQSP (SEQ ID NO: 47), is inserted at the C-terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 126.
  • An example of such mutant IL-2 is the mutant 2-168.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs as compared to SEQ ID NO: 1 or variant thereof, wherein one motif: is inserted by insertion of a Q residue at the T41 position by reference to SEQ ID NO: 1 (z.e.
  • LLQ LLQFKF
  • SEQ ID NO: 10 LLQFKF
  • mutant IL-2 comprises a sequence comprising or consisting of LQS, preferably a sequence comprising or consisting of GLQSP (SEQ ID NO: 48), at the C- terminus), optionally wherein the mutant IL-2 comprises, by reference to SEQ ID NO: 1, (i) a substitution of the cysteine residue 125 with a serine residue (z.e., C125S mutation), (ii) a substitution of the glutamine 74 residue with a glutamate residue (i.e., Q74E mutation), or (iii) both a substitution of the cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and a substitution of the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue i.e., a Q74E mutation).
  • the sequence comprising or consisting of LQS preferably the sequence comprising or consisting of GLQSP (SEQ ID NO: 48), is inserted at the C- terminus of the sequence with SEQ ID NO: 1.
  • the mutant IL-2 comprises or consists of the sequence with SEQ ID NO: 127.
  • An example of such mutant IL-2 is the mutant 2-169.
  • the present invention further relates to a method for incorporating at least one non-endogenous transglutaminase motif in human IL-2 with the sequence SEQ ID NO: 1 or from a variant thereof, comprising the steps of: inserting a Q residue at one or more of the following positions by reference to SEQ ID NO: 1: H16, L18, L19, N26, K32, N33, T41, F42, T51, L53, Q57, H79, E95, E100, E110 and S130, and/or inserting a motif at the N-terminus or C-terminus, wherein said motif comprises a sequence selected from the group comprising or consisting of LQS and TQ
  • cysteine residue 125 substituted with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation),
  • cysteine residue 125 with any other amino acid residue, preferably a serine residue (i.e., a C125S mutation) and substituting the glutamine 74 residue with any other amino acid residue, preferably a glutamate residue (i.e., a Q74E mutation).
  • the method further comprises a step of substituting the phenylalanine 42 residue with any other amino acid residue, preferably an alanine residue (i.e., F42A mutation), by reference to SEQ ID NO: 1.
  • any other amino acid residue preferably an alanine residue (i.e., F42A mutation)
  • the method comprises the insertion of at least two non- endogenous transglutaminase motifs.
  • one of the non-endogenous transglutaminase motifs comprises the insertion of a Q residue at the T41 or F42 position as reference to SEQ ID NO: 1.
  • the present invention further relates to a fusion protein comprising a mutant IL- 2 as defined hereinabove.
  • the fusion protein comprises at least one masking moiety, wherein the at least one masking moiety is linked to a Q residue of the at least one non- endogenous transglutaminase motif of the mutant IL-2 as defined hereinabove.
  • the mutant IL-2 comprises at least two non-endogenous transglutaminase motifs
  • the fusion protein comprises at least two masking moieties, wherein the at least two masking moieties are linked to a Q residue of each non- endogenous transglutaminase motif of the mutant IL-2 as defined hereinabove.
  • the fusion protein when the fusion protein comprises at least two masking moieties, the at least two masking moieties are different. In one embodiment, when the fusion protein comprises at least two masking moieties, the at least two masking moieties are similar.
  • the masking moiety refers to a moiety that, when linked to a Q residue of the non-endogenous transglutaminase motif of the mutant IL-2, prevents or reduces the signaling of the mutant IL-2, or the binding or affinity of the mutant IL-2 to its receptor.
  • Methods for measuring the IL-2 signaling are well known in the art and comprise, for example, the measurement of the induction of IL-2 receptor signaling (e.g., by detection of phosphorylated STAT5a), the measurement of the induction of T cell proliferation (e.g., by detection of Ki-67 using in particular CellTraceTM Cell Proliferation Kits, by direct assessment of T cell proliferation in the presence of IL-2, in T cell activation assay experiments (comprising, for example, the activation of cells with CD3 and CD28 in the presence of IL-2), or using cell lines that depend on IL-2 to proliferate, such as, for example CTLL2 cell line) and/or the measurement of an up-regulation of expression of activation markers (such as e.g., CD25, CD69, cytotoxic molecules, such as, for example, granzyme B, and the like).
  • activation markers such as e.g., CD25, CD69, cytotoxic molecules, such as, for example, granzyme B,
  • Affinity of a protein is commonly expressed as an equilibrium dissociation constant (KD).
  • KD equilibrium dissociation constant
  • the binding kinetics, such as the KD, of the at mutant IL-2 to its receptor are determined by surface plasmon resonance (SPR, e.g. , using BIAcore®).
  • SPR surface plasmon resonance
  • Other methods for measuring binding or affinity of proteins are well known in the art, and include, for example, cell-binding assays, such as competitive binding assays.
  • the mutant IL-2 comprised in the fusion protein as defined hereinabove has a reduced binding or affinity for its receptor as compared to the mutant IL-2 that is free (z.e., not associated with a masking moiety). In one embodiment, the mutant IL-2 comprised in the fusion protein as defined hereinabove has a reduced binding or affinity for 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 as compared to the mutant IL-2 that is free (z.e., not associated with a masking moiety).
  • the mutant IL-2 comprised in the fusion protein as defined hereinabove has a reduced binding or affinity for the IL-2R alpha, beta and/or gamma chain as compared to the mutant IL-2 that is free (z.e., not associated with a masking moiety).
  • the mutant IL-2 comprised in the fusion protein as defined hereinabove has a reduced binding or affinity for the IL-2R alpha, beta and/or gamma chain by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more as compared to the mutant IL-2 that is free (z.e., not associated with a masking moiety).
  • the mutant IL-2 comprised in the fusion protein as defined hereinabove has a reduced binding or affinity for the low-affinity, intermediate-affinity, and/or high-affinity IL-2 receptor as compared to the mutant IL-2 that is free (z.e., not associated with a masking moiety).
  • the mutant IL-2 comprised in the fusion protein as defined hereinabove has a reduced binding or affinity for the low- affinity, intermediate-affinity, and/or high-affinity IL-2 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 as compared to the mutant IL-2 that is free (z.e., not associated with a masking moiety).
  • the at least one masking moiety may be any type of molecule.
  • the at least one masking moiety is selected from the group comprising or consisting of glycans, lipids, proteins, peptides, small organic molecule and polymers.
  • the at least one masking moiety is a glycan.
  • the at least one masking moiety is a polymer, preferably a water-soluble polymer.
  • the water-soluble polymer is selected from the group comprising or consisting of polyethylene glycol (PEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxy ethylated polyol), poly(olefinic alcohol), poly (vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly (hydroxy alkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly (N- acryloylmorpholine), and combinations thereof.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • copolymers of ethylene glycol and propylene glycol poly(oxy ethylated polyol), poly(olefinic alcohol), poly (vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly (hydroxy alkylmethacrylate), poly(sacchari
  • the water-soluble polymer is a polyethylene glycol (PEG).
  • the at least one masking moiety is a linear PEG.
  • a linear PEG refers to a PEG composed of one linear chain.
  • the at least one masking moiety is a linear PEG up to about 20kDa, preferably of about 10 kDa.
  • the at least one masking moiety is a branched PEG.
  • a branched PEG refers to a PEG composed of at least two linear PEGs that are linked together.
  • An example of a branched PEG is a Y-shaped PEGs, such as, for example, a Y-shaped PEG with two PEGs of about 20 kDa.
  • the at least one masking moiety is a PEG having a weight comprised from about 5 to about 50 kDa, preferably from about 10 to about 40 kDa.
  • the fusion protein when the fusion protein comprises at least two masking moieties, the at least two masking moieties may be PEG that are similar or different.
  • the fusion protein further comprises at least one binding moiety, such as, for example, antibodies or fragments thereof, or antibody mimetics.
  • a binding moiety refers to any molecule capable of binding to a target.
  • the binding moiety and the masking moiety are one single molecule, i.e. the single molecule is able to act as both a masking moiety and a binding moiety.
  • the fusion protein further comprises at least one antibody or antigen-binding fragment thereof.
  • the at least one antibody or antigen-binding fragment thereof comprised in the fusion protein is selected from the group comprising or consisting of a whole antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a defucosylated antibody, a bispecific antibody, a diabody, a triabody, a tetrabody, a nanobody, a domain antibody, and a unibody, preferably the at least one antibody or antigen-binding fragment is a Fab.
  • the at least one antibody or antigen-binding fragment thereof is polyclonal. In one embodiment, the at least one antibody or antigen-binding fragment thereof is monoclonal.
  • antibodies or antigen-binding fragments thereof comprised in a fusion protein according to the present invention can be modified using known methods.
  • Modifications can also have been made to framework residues within the heavy chain variable region (VH) and/or the light chain variable region (VL), e.g., to improve the properties of the antibody or fragment.
  • framework modifications are made to decrease the immunogenicity of the antibody or fragment.
  • one approach is to “back-mutate” one or more framework residues to the corresponding germline sequence.
  • antibodies or antigen-binding fragments thereof can be aglycosyled (z.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 alter the antibody-dependent cellular cytotoxicity activity of the antibody or fragment thereof. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • the fusion protein further comprises at least one antibody mimetic.
  • antibody mimetic include, without limitation, affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody
  • the at least one antibody or antigen-binding fragment or the at least one antibody mimetic targets an antigen, such as, for example, a cancer antigen or an immune checkpoint protein.
  • cancer antigens include, without limitation, EGFR, HER2, HER3, HER 4, NY-ESO -1, GPC -3, CLL -1, BCMA, GD2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCL27, CCL28, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6), Mucin family (MUC1, MUC2 MUC3 A, MUC3 B, MUC4, MUC5 AC, MUC5 B, MUC6, MUC7, MUC8, MUC12, MUC6, MUCUNA, MUC16, MUC17, MUCUNA, MUCIN, TERT, TLR, Survivin, CD 123, TIGIT, DLL3, PSMA, Fibroblast Growth Factor Receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), nerve growth factor receptor (NG
  • immune checkpoint proteins include, without limitation, PD-1, PD- Ll, CTLA-4, LAG-3, 0X40, CD28, CD40, CD47, CD70, CD80, CD122, GTIR, A2AR, B7-H3 (CD276), B7-H4, IDO, KIR, TIM -3 or 4-1 BB (CD137).
  • the at least one antibody or antigen-binding fragment or antibody mimetic targets HER2.
  • HER2 human epidermal growth factor receptor 2, also known as CD340 (cluster of differentiation 340)
  • CD340 cluster of differentiation 340
  • the at least one antibody or antigen-binding fragment or antibody mimetic targets PD-1.
  • PD-1 Programmed cell death protein 1, also known as CD279 (cluster of differentiation 279)
  • CD279 cluster of differentiation 279
  • the fusion protein comprises at least one mutant IL-2, at least one masking moiety and at least one binding moiety, preferably an antibody or antigenbinding fragment thereof or an antibody mimetic, wherein the at least one masking moiety is linked to the at least one mutant IL-2 with a linker and wherein the at least one mutant IL-2 is linked to the at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, with another linker.
  • the linkers linking (i) the at least one masking moiety to the at least one mutant IL-2 and (ii) the at least one mutant IL-2 to the at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic are similar. In one embodiment, the linkers linking (i) the at least one masking moiety to the at least one mutant IL-2 and (ii) the at least one mutant IL-2 to the at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, are different.
  • the fusion protein comprises at least one mutant IL-2, at least two masking moieties and at least one binding moiety, preferably an antibody or antigenbinding fragment thereof or an antibody mimetic, wherein the at least two masking moieties are linked to the at least one mutant IL-2 with a linker.
  • the linkers linking the at least two masking moieties to the at least one mutant IL-2 are similar. In one embodiment, the linkers linking the at least two masking moieties to the at least one mutant IL-2 are different. [0456] In one embodiment, the linker linking the at least one mutant IL-2 to the at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, is a cleavable linker. In another embodiment, the linker linking the at least one mutant IL-2 to the at least one binding moiety, preferably an antibody or antigenbinding fragment thereof or an antibody mimetic, is an uncleavable linker.
  • linker z.e., cleavable or uncleavable
  • linker z.e., cleavable or uncleavable
  • a releasable IL-2 or an IL-2 irreversibly linked to the binding moiety preferably an antibody or antigen-binding fragment thereof or an antibody mimetic.
  • the linker linking the at least one masking moiety to the at least one mutant IL-2 is a cleavable linker.
  • the linker linking the at least one masking moiety to the at least one mutant IL-2 is an uncleavable linker. It will be understood that the nature of the linker (z.e., cleavable or uncleavable) will depend on the therapeutic applications, since, depending on the conditions, it may be desirable to have either a releasable masking moiety or an IL-2 irreversibly linked to the masking moiety.
  • the fusion protein comprises at least one or at least two masking moiety(ies), at least one mutant IL-2 and at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, wherein said elements are linked by cleavable linkers.
  • the fusion protein comprises at least one or at least two masking moiety(ies), at least one mutant IL-2 and at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, wherein said elements are linked by uncleavable linkers.
  • the fusion protein comprises at least one or at least two masking moiety(ies), at least one mutant IL-2 and at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, wherein the at least one or at least two masking moiety(ies) are linked to the at least one mutant IL-2 with an uncleavable linker, and wherein the at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, is linked to the at least one mutant IL-2 with a cleavable linker.
  • the fusion protein comprises at least one or at least two masking moiety(ies), at least one mutant IL-2 and at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, wherein the at least one or at least two masking moiety(ies) are linked to the at least one mutant IL- 2 with a cleavable linker, and wherein the at least one binding moiety, preferably an antibody or antigen-binding fragment thereof or an antibody mimetic, is linked to the at least one mutant IL-2 with an uncleavable linker.
  • the uncleavable linker is a peptide having a length ranging from 2 and 10 amino acids.
  • a glycine- serine doublet provides a particularly suitable hinge domain (GS linker).
  • the hinge domain is a Gly/Ser linker.
  • Gly/Ser linkers include, but are not limited to, GS linkers, G2S linkers, G3S linkers, G4S linkers.
  • G2S linkers include, but are not limited to, GGS.
  • linkers A4T (SEQ ID NO: 129) may be also used in the context of the present invention.
  • the cleavable linker is a cleavable peptide having a length ranging from 2 and 10 amino acids. [0469] In one embodiment, the cleavable linker is cleavable by enzymes, photoirradiation, pH, or chemical agents.
  • the cleavable linker is cleavable by enzymes, such as, for example, linkers cleavable by proteases, linkers cleavable by beta glucuronidase or peptide linkers.
  • the cleavable linker is cleavable by pH.
  • linker cleavable by pH include, without limitation, hydrazine linker, maleic acid amide linker or cis-aconityl linker.
  • 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.
  • the linker between the binding moiety and the IL-2 mutant may or may not be proteolytically cleavable, and the linker between the masking moiety and the IL-2 may or may not be cleavable.
  • Another object of the invention is an isolated nucleic acid encoding a mutant IL- -2 or a fusion protein according to the present invention.
  • an “isolated nucleic acid”, as used herein, is intended to refer to a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • the term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
  • nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as for example plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • a suitable vector such as for example plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • another object of the present invention is an expression vector comprising a nucleic acid encoding a mutant IL-2 or a fusion protein according to the present invention.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform a host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said fusion protein upon administration to a host.
  • promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like.
  • Any expression vector for animal cell can be used, so long as a gene encoding the fusion protein can be inserted and expressed.
  • suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4 and the like.
  • Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
  • viral vector include adenoviral, retroviral, herpes virus and AAV vectors.
  • Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, Gpenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found in the art.
  • Another object of the invention is an isolated host cell comprising said vector.
  • Said host cell may be used for the recombinant production of a mutant IL-2 or a fusion protein according to the present invention.
  • host cells may be prokaryote, yeast, or eukaryote cells, preferably mammalian cells, such as, for example: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby 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.
  • mammalian cells such as, for example: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (19
  • Another object of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of or consisting of at least one mutant IL-2, at least one fusion protein, at least one nucleic acid, or a at least one expression vector according to the present invention, and at least one pharmaceutically acceptable excipient.
  • compositions of the present invention examples include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, poly acrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium
  • Another object of the present invention is a medicament comprising, consisting essentially of or consisting of at least one mutant IL-2, at least one fusion protein, at least one nucleic acid, or at least one expression vector according to the present invention.
  • 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 manufacture of a medicament for treating a disease, disorder or symptoms in a subject in need thereof.
  • compositions, pharmaceutical composition or medicament for use in administration to a subject, will be formulated for administration to the subject.
  • composition, pharmaceutical composition or medicament according to the present invention is administered (or is to be administered) parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir.
  • the composition, pharmaceutical composition or medicament is administered (or is to be administered) by injection, including, without limitation, subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection, or infusion techniques.
  • Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms, and the like.
  • Sterile injectable forms of the compositions, pharmaceutical compositions or medicaments of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxy ethylated versions.
  • oils such as olive oil or castor oil
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • a long-chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • the mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is to be administered to the subject in need thereof in a therapeutically effective amount.
  • the total daily usage of the IL-2 mutant, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; activity of the IL-2 mutant, fusion protein, nucleic acid, or expression vector employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific IL-2 mutant, fusion protein, nucleic acid, or expression vector employed; the duration of the treatment; drugs used in combination or coincidental with the specific IL-2 mutant, fusion protein, nucleic acid, or expression vector employed; and like factors well known in the medical arts.
  • the compound it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the present invention also relates to at least one mutant IL-2, fusion protein, nucleic acid or expression vector as described herein for use as a medicament, i.e., for treating (or for use in treating) diseases, disorders or symptoms in a subject in need thereof.
  • 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 a subject in need thereof, thereby treating diseases, disorders or symptoms in the subject.
  • the present invention also relates to methods for treating diseases, disorders or symptoms or methods for promoting Treg cell expansion in a subject in need thereof, comprising administering to the subject the at least one mutant IL-2, fusion protein, nucleic acid or expression vector as described herein.
  • diseases that may be treated in the present invention, include, but are not limited to, cancers, and inflammatory diseases.
  • the disease, disorder or symptom to be treated is cancer.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes, without limitation, diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • cancer further encompasses both primary and metastatic cancers.
  • cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, endometrial, pancreas or uterus.
  • the cancer may be selected in the following non-limiting list: malignant neoplasm; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma associated with familial polyposis coli; solid carcinoma; malignant carcinoid tumor; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic a
  • the disease, disorder or symptom to be treated is an inflammatory disease.
  • inflammatory diseases include, but are not limited to, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, arthritis uratica, gout, chronic polyarthritis, periarthritis humeroscapularis, cervical arthritis, lumbosacral arthritis, enteropathic arthritis and ankylosing spondylitis, asthma, dermatitis, psoriasis, scleroderma, polymyositis, dermatomyositis, juvenila dermatomyositis, primary biliary cirrhosis, fibrosis, cystic fibrosis, pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, dediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephro
  • mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used alone.
  • mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with at least one further therapeutic agent.
  • the at least one further therapeutic agent and the mutant IL-2, fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention are administered as one composition or as separate compositions, as appropriate.
  • additional therapeutic agents include, but are not limited to, chemotherapeutic agents, targeted cancer therapy, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti- angiogenic agents, cell cycle control/apoptosis regulating agents, hormonal regulating agents, tyrosine kinase inhibitors (TIKs), vaccines, immune checkpoint inhibitors, CAR- T and CAR-NK cell therapies, CAR macrophage therapy, tumor-infiltrating lymphocytes (TIL) therapies, immune engaging biologies, steroids, JAK inhibitors, and other immunosuppressive and/or anti-inflammatory drugs including corticoids, such as, for example, glucocorticoids.
  • corticoids such as, for example, glucocorticoids.
  • the at least one further therapeutic agent is a therapeutic agent useful for treating the specific disease, disorder or condition to be treated in the present invention.
  • the at least one further therapeutic agent may be selected from the group comprising, but not limited to, chemotherapeutic agents, targeted cancer therapy, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti- angiogenic agents, cell cycle control/apoptosis regulating agents, hormonal regulating agents, tyrosine kinase inhibitors (TIKs), vaccines, immune checkpoint inhibitors, CAR-T and CAR-NK cell therapies, CAR macrophage therapy, tumor-infiltrating lymphocytes (TIL) therapies, immune engaging biologies, and other immunosuppressive and/or anti-inflammatory drugs selected from corticoids, such as, for example, glucocorticoids.
  • corticoids such as, for example, glucocorticoids.
  • the at least one further therapeutic agent may be selected from the group comprising, but not limited to, antiinflammatory drugs, steroids, and JAK inhibitors.
  • Figure 1 presents the results with the fusion proteins comprising 2-102, 2-105 to 2-107, 2-112 to 2-116, and 2-120 IL-2 mutants.
  • Figure IB presents the results with the fusion proteins comprising 2-121 to 2-126, 2-130 and 2-131 IL-2 mutants.
  • Figure 2 is a combination of two graphs showing the percentage of pSTAT5 expression on Treg cells (A) and on CD8 T cells (B) induced by the following fusion proteins: IL2-Fab (z.e. a wild type IL-2 fused to a Fab against HER2, used as a control), and the fusion proteins comprising either the 2-114 or the 2-115 IL-2 mutant, a 10 kDa PEG fused to the non-endogenous transglutaminase motif of IL-2 and a Fab against HER2 (i.e. fusion proteins 2-114-P10 and 2-115-P10, respectively).
  • IL2-Fab z.e. a wild type IL-2 fused to a Fab against HER2, used as a control
  • the fusion proteins comprising either the 2-114 or the 2-115 IL-2 mutant, a 10 kDa P
  • Figure 3 is a combination of 4 graphs showing the percentage of pSTAT5 expression on CD4 T cells, CD8 T cells, Treg cells and NK cells induced by a fusion protein comprising the 2-120 IL-2 mutant, a 10 kDa PEG fused to the non-endogenous transglutaminase motif of IL-2 and a Fab against HER2 (2-120-P10).
  • the continuous line represents 2-120 and the dashed line represents the construct 2-120-P10.
  • Figure 4 is a combination of 4 graphs showing the percentage of pSTAT5 expression on CD4 T cells, CD8 T cells, Treg cells and NK cells induced by a fusion protein comprising the 2-121 IL-2 mutant, a 10 kDa PEG fused to the non-endogenous transglutaminase motif of IL-2 and a Fab against HER2 (2-121-P10).
  • the continuous line represents 2-121 and the dashed line represents the construct 2-121-P10.
  • Figure 5 is a combination of 4 graphs showing the percentage of pSTAT5 expression on CD4 T cells, CD8 T cells, Treg cells and NK cells induced by the fusion protein comprising the 2-122 IL-2 mutant, a 10 kDa PEG fused to the non-endogenous transglutaminase motif of IL-2 and a Fab against HER2 (2-122-P10).
  • the continuous line represents 2-122 and the dashed line represents the construct 2-122-P10.
  • Figure 6 is a combination of 4 graphs showing the percentage of pSTAT5 expression on CD4 T cells, CD8 T cells, Treg cells and NK cells induced by the fusion protein comprising the 2-123 IL-2 mutant, a 10 kDa PEG fused to the non-endogenous transglutaminase motif of IL-2 and a Fab against HER2 (2-123-P10).
  • the continuous line represents 2-123 and the dashed line represents the construct 2-123-P10.
  • Figure 7 is a combination of 4 graphs showing the percentage of pSTAT5 expression on CD4 T cells, CD8 T cells, Treg cells and NK cells induced by the fusion protein comprising the 2-124 IL-2 mutant, a 10 kDa PEG fused to the non-endogenous transglutaminase motif of IL-2 and a Fab against HER2 (2-124-P10).
  • the continuous line represents 2-124 and the dashed line represents the construct 2-124-P10.
  • Figure 8 is a combination of 7 graphs showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by fusion proteins according to the present invention.
  • % STAT5 is normalized against IL2-Fab. It presents the effects of IL2-Fab (control); and of the following fusion proteins: a fusion protein comprising the 2-114 IL- 2 mutant, a Fab against HER2 and with or without a 10 kDa PEG (2-114 and 2-114-P10, respectively) (A), a fusion protein comprising the 2-115 IL-2 mutant, a Fab against HER2 and with or without a 10 kDa PEG (2-115 and 2-115-P10, respectively) (B), a fusion protein comprising the 2-120 IL-2 mutant, a Fab against HER2 and with or without a 10 kDa PEG ( 2-120 and 2-120-P10, respectively) (C), a fusion protein comprising the 2- 121 IL-2 mutant, a Fab against Fab
  • a mutant IL-2 according to the present invention i.e. 2-132 to 2-135, 2-137, 2-138 and 2-140 IL-2 mutant
  • Figure 10A presents the results with the fusion proteins comprising 2-142 to 2-147, and 2-150 to 2-153 IL-2 mutants.
  • Figure 10B presents the results with the fusion proteins comprising 2-154 to 2- 161 IL-2 mutants.
  • Figure 12 is a combination of 3 graphs showing the percentage of pSTAT5 expression on Treg cells induced by IL2-Fab (control); and by the following fusion proteins according to the present invention: a fusion protein comprising the 2-130 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-130-P10 and 2-130, respectively) (A), a fusion protein comprising the 2-135 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-135-P10 and 2-135, respectively) (B), a fusion protein comprising the 2-137 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-137-P10 and 2-137, respectively) (C).
  • Unstimulated represents cells which have not been activated.
  • Figure 13 is a combination of 3 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 present invention: a fusion protein comprising the 2-130 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-130-P10 and 2-130, respectively) (A), a fusion protein comprising the 2-135 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-135-P10 and 2-135, respectively) (B), a fusion protein comprising the 2-137 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-137-P10 and 2-137, respectively) (C).
  • A a fusion protein comprising the 2-130 IL-2 mutant, a Lab against HER2 and with or without a 10 kDa PEG (2-130-P10 and 2-130, respectively)
  • B
  • FIG. 14 is a schema showing 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 moiety, and may or may not have a linker between the binding moiety and the IL-2 mutant.
  • the linker between the binding moiety and the IL-2 mutant may or may not be proteolytically cleavable, and the linker between the masking moiety and the IL-2 mutant may or may not be cleavable.
  • Figure 15 is a combination of 4 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 a fusion protein comprising the 2-130 IL-2 mutant, a Fab against HER2 and with or without a 40 kDa PEG (2-130-P40 and 2-130, respectively).
  • Unstimulated represents cells which have not been activated.
  • Figure 16 is a combination of 4 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 a fusion protein comprising the 2-132 IL-2 mutant, a Fab against HER2 and with or without a 40 kDa PEG (2-132-P40 and 2-132, respectively).
  • Unstimulated represents cells which have not been activated.
  • Figure 17 is a combination of 3 graphs showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by IL2-Fab (control); and by a fusion protein comprising the 2-135 IL-2 mutant, a Fab against PD-1 and with or without a 40 kDa PEG (2-135-P40 and 2-135, respectively) (A), a fusion protein comprising the 2-136 IL-2 mutant, a Fab against PD-1 and with or without a 40 kDa PEG (2-136-P40 and 2- 136, respectively) (B), a fusion protein comprising the 2-137 IL-2 mutant, a Fab against PD-1 and with or without a 40 kDa PEG (2-137-P40 and 2-137, respectively).
  • % STAT5 is normalized against IL2-Fab.
  • Figure 18 is a graph showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by IL2-Fab (control); and by a fusion protein comprising the 2-210 IL-2 mutant, a Fab against HER2 and with or without a 40 kDa PEG (2-210-P40 and 2- 210, respectively). % STAT5 is normalized against IL2-Fab.
  • Figure 19 is a graph showing in vivo PK study with control fusion proteins (IL2- Fab with a F42 mutation (non-alpha IL2-Fab) or without F42 mutation (WT IL2-Fab), or a fusion protein comprising the 2-132 mutant, a Fab against PD-1 and with a 10 kDa or a 40 kDa PEG.
  • control fusion proteins IL2- Fab with a F42 mutation (non-alpha IL2-Fab) or without F42 mutation (WT IL2-Fab
  • a fusion protein comprising the 2-132 mutant, a Fab against PD-1 and with a 10 kDa or a 40 kDa PEG.
  • Figure 20 is a graph showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by IL2-Fab (control); and by a fusion protein comprising the l ' -I' l IL-2 mutant, a Fab against PD-1 and with or without a 40 kDa PEG (2-222-P40 and 2- 222, respectively). % STAT5 is normalized against IL2-Fab.
  • Figure 21 is a graph showing the percentage of pSTAT5 expression on NK cells (NK92 cells) induced by IL2-Fab (control); and by a fusion protein comprising the 2-138 IL-2 mutant, a Fab against PD-1 and with or without a 40 kDa PEG (2-138-P40 and 2- 138, respectively). % STAT5 is normalized against IL2-Fab.
  • DNA coding for the amino acid sequence of the listed constructs were synthesized and cloned into the mammalian transient expression vector plasmid pETE V2. All constructs were expressed using CHO based transient expression system and the resulting antibody containing cell culture supernatants were clarified by centrifugation and filtration. All constructs were purified from cell culture supernatants via affinity chromatography. Purified antibody was buffer exchanged into phosphate buffered saline solution. The purity of the antibodies was determined by reducing and denaturing Sodium Dodecyl Sulfate Polyacrylamide gels and analyzed by semi-preparative SEC. Antibody concentrations were determined by measuring absorbance at 280nm and calculated theoretical extinction coefficient. pSTAT5 activation assay on hPBMC
  • PBMC peripheral blood mononuclear cells
  • the plate was centrifuged at 400g for 2min prior to adding 50ul/well of intercellular staining antibody (pSTAT5-AF647-FL6). The plate was incubated at RT for 30min in dark then washed once with 150ul R4 buffer wash reagent. After washing each sample was resuspended in 150ul of buffer R4 and transferred to a microtitre tube and read on the Navios EX.
  • a capture assay set up was utilised for receptor binding analysis.
  • Anti-human IgG (Cat No. BR100839) was coupled to CM5 chips (GE Healthcare) using standard amine chemistry to ⁇ 9000RU.
  • Human IL-2Roc-Fc at I pg/ml in HBS-P+ running buffer was captured to -60RU on flow cell 2.
  • Human IL-2Ry-Fc at I pg/ml in HBS-P+ running buffer was captured to -120RU on flow cell 1.
  • Her2 binding of the Fab portion was assessed in the same assay set up with Her2-Fc captured on the chip and analyte run over the surface.
  • a Nunc MaxiSorp was coated with 25 ul/well of anti-Nivolumab Fab antibody (BioRad- HCA299 Human Fab) protein at 2 pg/ml in coating buffer (PBS lx). 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 3 x PBS-0.05% Tween-20. Serum samples collected from test groups were prepared and added to the plate prior to incubation at room temperature for 2 hours and subsequent washing 3 x PBS-0.05% Tween-20.
  • Detection antibody anti- kappa light chain HRP antibody (Novus NB500- 331H) was added to the plate with incubation and washing steps as detailed above. This was followed by the addition of the detection sustrate (TMB) and optical absorbance measured at 450nm.
  • TMB detection sustrate
  • Example 1 Mutant IL-2 with at least one non-endogenous transglutaminase motif
  • Fusion proteins comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif and a Fab against HER2, were constructed and studied regarding different parameters: i) their ability to be produced and purified, ii) their ability to bind HER2, CD25 and CD 122, iii) their ability to activate CD4 T cells, Treg cells, CD8 T cells and NK cells, and iv) their ability to be bio-conjugated with a range of PEG moieties of varying sizes and structures including, but not limited to, lOkDa linear and 40kDa branched. Results are disclosed in the Figure 1.
  • All the constructs comprising a mutant IL-2 having at least one non-endogenous transglutaminase motif i) can be robustly produced and purified, ii) bind to HER2 and CD 122 (IL-2RP), iii) are able to activate CD4 T cells, CD8 T cells, and NK cells, and iv) are able to be bio-conjugated with a PEG.
  • All the fusion proteins except the ones comprising 2-106, 2-107, 2-130 and 2-131 IL-2 mutants are able to bind to CD25 (IL- 2Ra), while the fusion proteins comprising 2-106, 2-107, 2-130 and 2-131 IL-2 mutants do not bind to CD25 and therefore exhibit reduced potency in activation of Treg cells.
  • fusion proteins comprising a mutant IL-2 with at least one non- endogenous transglutaminase motif as described herein, a PEG fused to the IL-2 mutant by transglutamination with a linker, and a Fab against HER2 were also generated, and their ability to activate Treg cells, NK cells and CD8 T cells was evaluated.
  • the presence of a 10 kDa PEG fused to the 2-114 or the 2-115 IL-2 mutant prevents the activation of CD8 T cells and has a lower impact on Treg cell activation. The same results were obtained with the 2-125 and the 2-126 mutants (data not shown).
  • Figures 3-7 show that the presence of a PEG fused to the 2-120, 2-121, 2-122, 2-123 or 2-124 IL-2 mutant prevents the activation of CD4 T cells, and CD8 T cells, and has a lower impact on activation of NK cells and Treg cells.
  • Figure 8 further shows that the presence of a PEG fused to the 2-114, 2-115, 2-120, 2-121, 2-124, 2-125 or the 2-126 IL-2 mutant impact NK cells activation to a different extent depending on the IL-2 mutant.
  • the presence of a 10 kDa PEG fused to this IL-2 mutant strongly prevents the activation of both CD8 T cells and Treg cells, and the presence of a 40 kDa PEG prevents the activation of CD4 and CD8 T cells as well as the activation of Treg cells and NK cells.
  • Figure 18 shows that the presence of a 40kDa PEG fused to the 2-210 IL-2 mutant impacts NK cells activation.
  • a fusion protein comprising a mutant IL-2 having a PEG fused to a non-endogenous transglutaminase motif prevents the activation of CD4 T cells and CD8 T cells, and, depending on the IL-2 mutant comprised in the fusion protein, impact NK cells and Treg cells activation to a different extent.
  • Fusion proteins comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif and a F42A mutation, fused to a Fab against HER2, were constructed and studied regarding different parameters: i) their ability to be produced and purified, ii) their ability to bind HER2, CD25 and CD 122, iii) their ability to activate CD4 T cells, CD8 T cells, Treg cells and NK cells, and iv) their ability to be bio-conjugated with a PEG. Results are disclosed in the Figure 9.
  • All the constructs comprising IL-2 mutants having at least one non-endogenous transglutaminase motif and a F42A mutation i) can be robustly produced and purified, ii) bind to HER2 and CD 122 but not to CD25, iii) are able to activate CD4 T cells, CD8 T cells, Treg cells and NK cells, and iv) are able to be bio-conjugated with a PEG.
  • fusion proteins comprising a mutant IL-2 with at least one non- endogenous transglutaminase motif and a F42A mutation, a PEG fused to the IL-2 mutant by transglutamination with a linker, and a Fab against HER2 were also generated, and their ability to activate Treg cells and CD8 T cells was evaluated.
  • the presence of a 10 kDa PEG fused to the 2-135 or the 2-137 IL-2 mutant prevents the activation of CD8 T cells and, depending on the IL-2 mutant, impacts Treg cells activation to a different extent.
  • Fusion proteins comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif and a F42A mutation, a 40 kDa PEG fused to the IL-2 mutant by transglutamination with a linker, and a Fab against PD-1 were also generated, and their impact on NK cells activation was evaluated.
  • the presence of a 40 kDa PEG fused to the 2-135, 2-136, 2-137, 2-138 or the l ' -I' l mutant affects NK cells activation.
  • fusion proteins comprising a mutant IL-2 with at least one non-endogenous transglutaminase motif and a F42A mutation (i.e. 2-132 mutant), a 10 kDA or 40 kDa PEG fused to the IL-2 mutant by transglutamination with a linker, and a Fab against PD-1.
  • the fusion protein with a branched 40kD PEG has an improved PK over the fusion protein with a linear lOkD PEG.
  • both masked fusion proteins demonstrate improved PK over the control fusion proteins.
  • a fusion protein comprising a mutant IL-2 having a PEG fused to a non-endogenous transglutaminase motif and a F42A mutation prevents the activation of CD8 T and CD4 T cells, and, depending on the IL-2 mutant comprised in the fusion protein, impact Treg cells and NK cells activation to a different extent.
  • Example 3 Mutant IL-2 with at least two non-endogenous transglutaminase motifs
  • Fusion proteins comprising a mutant IL-2 with at least two non-endogenous transglutaminase motifs, fused to a Fab against HER2, were constructed and studied regarding different parameters: i) their ability to be produced and purified, ii) their ability to bind HER2, CD25 and CD 122, and iii) their ability to activate CD4 T cells, Treg cells and NK cells. Results are disclosed in the Figures 10A and 10B.
  • All the constructs comprising IL-2 mutants having at least two non-endogenous transglutaminase motifs i) can be robustly produced and purified, ii) bind to HER2 and CD 122 but not to CD25, and iii) are able to activate CD4 T cells, CD8 T cells, Treg cells and NK cells.
  • Fusion proteins comprising a mutant IE-2 with at least two non-endogenous transglutaminase motifs with one of the motifs being the sequence EEQFKF (SEQ ID NO: 10) with a Q41 residue, fused to a Fab against HER2, were constructed and studied regarding different parameters: i) their ability to be produced and purified, ii) their ability to bind HER2, CD25 and CD 122, iii) their ability to activate CD4 T cells, Treg cells and NK cells, and iv) their ability to be bio-conjugated with a PEG. Results are disclosed in the Figure 11.
  • All the constructs comprising IL-2 mutants having at least two non-endogenous transglutaminase motifs with one of the motifs being the sequence LLQFKF (SEQ ID NO: 10) with a Q41 residue i) can be robustly produced and purified, ii) bind to HER2 and CD 122 and CD25, iii) are able to activate CD4 T cells, CD8 T cells and NK cells, but not Treg cells, and iv) are able to be bio-conjugated with a PEG (among the ones tested).

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Abstract

La présente invention concerne des variants mutationnels de la protéine IL-2 qui facilitent la conjugaison chimique et leur utilisation potentielle en tant que traitements contre le cancer ou une maladie inflammatoire.
PCT/EP2024/073705 2023-08-23 2024-08-23 Protéines variantes d'interleukine-2 qui facilitent la conjugaison chimique covalente et leurs utilisations Pending WO2025040797A1 (fr)

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