IL-15 MUTEINS WITH PH-DEPENDENT BINDING FOR IL-15Rbeta

Abstract

The present invention describes a mutein of IL-15 that can bind to its receptor subunit IL-15Rβ only at acidic pH. The mutant, called Switch-15β, has 8 amino acid changes at the interface with the receptor. The binding of Switch-15β to IL-15Rβ is disrupted at neutral pH but retained at low pH. The present claims are directed towards Interleukin-15 (IL-15) 's innovative use and pH-biased. The IL-15 mutein of the present invention is thus tailored to become more active or stable within the acidic conditions of the tumor microenvironment. This pH-switchable characteristic ensures that the therapeutic activity of IL-15 is maximized where it is most needed, enhancing its ability to stimulate immune cells such as natural killer (NK) cells and CD8+ T cells specifically within the tumor site. Such precision not only improves the efficacy of the treatment but also potentially reduces systemic side effects of IL-15 by limiting the activation of the immune system to the targeted area.

Description

[0001] IL-15 MUTEINS WITH PH-DEPENDENT BINDING FOR IL-15RP

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular immunology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Cytokines are powerful organic messengers of the immune system. They are secreted in response to stimuli and act locally to regulate a wide range of biological responses in different cell types, controlling every aspect of human health and diseases, including cancer and autoimmunity, allergy and the response to infection. Natural cytokines evolved to coordinate the correct functioning of the immune system. From activating T or NK cells to controlling inflammatory responses, cytokines influence countless cellular phenomena in vertebrate immunity.

[0006] Most cytokines are pleiotropic, meaning that they act across multiple cell types or upon diverse and often divergent immune processes1. This pleiotropy has proven to be a significant obstacle in developing cytokine therapeutics. Existing cytokine therapeutics, such as IL-2 and IL- 15, demonstrate meaningful efficacy in cancer and other diseases, but their applications are limited by significant on-target toxicity and off-target side effects2. Indeed, high-dose IL- 15 therapy toxic effects include high fever, neutropenia, thrombocytopenia, and hypotension which appears to be mediated in part by NK cells3. However, cytokines are critical in modulating immune function and their potential use as immunotherapy drugs cannot be overlooked. Furthermore, while native cytokines act locally, cytokine therapeutics are typically administered systemically, resulting in poor on-target efficacy and often inducing severe dose- related toxicities4. Overcoming these challenges represents a significant hurdle in developing next-generation cytokine therapeutics, emphasizing the urgent need for more specific, less toxic cytokine-based therapies.

[0007] Beyond immune modulation, IL- 15 has demonstrated important roles in skeletal muscle homeostasis and metabolism, promoting muscle protein synthesis while inhibiting protein degradation. Muscle cachexia, characterized by progressive loss of skeletal muscle mass and function, affects patients with cancer, chronic kidney disease, heart failure, COPD, and other chronic inflammatory diseases. Despite this recognized therapeutic potential, current treatment options for muscle cachexia remain limited, representing a significant unmet medical need (Carbo N et al., 2000, Br J Cancer & Quinn L.S et al., 2002, Exp Cell Res).

[0008] A key factor contributing to bioactivity and specificity by cytokines is the stability of cytokinecytokine receptor interactions5. Notably, while the ability of cytokines to stimulate a range of circulating immune cells has been extensively investigated under normal physiological conditions, how cytokines function by their extracellular microenvironment, including the tumour microenvironment (TME), has remained poorly elucidated6,7. Importantly, a high degree of fermentative glycolysis by tumour cells results in the accumulation of lactic acid and a low pH tumour microenvironment that contribute to immune escape and cancer progression8. Indeed, it is now well established that bound cytokine-receptor complexes are internalized and dissociate in the acidic environment of endosomes, such as that found within lymph nodes and inflammatory tissues such as the TME8,9.

[0009] We recently discovered a new mechanism by which the extracellular environment affects cytokine signalling and immune responses. We found that extracellular acidic pH (pHe), can profoundly impact signalling by IL-2 by preventing the salt bridge-mediated interaction with its high-affinity receptor with consequence for tumour immunity and immunotherapy10. We hypothesize that inactivation of potent T-cell stimulatory cytokines in acidic pHe is a major driver of immunotherapy resistance in tumours. Our research showed that the pH-dependency of cytokine-cytokine receptor interactions provides a basis for cytokine engineering and developed a conditional cytokine switch for therapeutic immunomodulation within the acidic microenvironment of tumours10.

[0010] SUMMARY OF THE INVENTION:

[0011] The present invention is defined by the claims. In particular, the present invention relates to IL- 15 muteins with pH-dependent binding for IL-15RP.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Main definitions:

[0014] As used herein, the term “about” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0015] As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0016] As used herein, the term “IL- 15” has its general meaning in the art and refers to a cytokine with structural similarity to IL-2 (GRABSTEIN et al., Science, vol. 264(5161), p:965-968, 1994). More particularly, IL-15 is a 14-15 kDa glycoprotein that is a member of the four-a-helix bundle cytokine family. IL- 15 has a 162 amino acid long open reading frame with a 48 amino acid long signal peptide and a 114 aa mature protein. An exemplary amino acid sequence for IL-15 is shown as SEQ ID NO:1. According to the present invention, the amino acid residue at position 49 in SEQ ID NO: 1 denotes the amino acid at position 1 in IL-15. Human IL-15 is encoded by a 34-kilobase locus at chromosome-4q31. The IL-15 gene comprises 9 exons and 8 introns, with 5 exons (exons 4-8) encoding the mature protein. Similar to IL-2, IL- 15 is able to trigger both proliferation of and immunoglobulin production by normal B lymphocytes. IL- 15 also stimulates the proliferation of NK cells and activated CD4+ and CD8+ T cells, and it facilitates the induction of cytolytic effector cells (such as lymphokine-activated killer cells). Finally, the numbers of CD8+ memory T cells are maintained in animals by a balance between the stimulatory effect of IL-15 and the suppressive effects of IL-12. Signaling pathway of IL- 15 begins with binding to IL-15Ra receptor, with subsequent presentation to surrounding cells bearing IL-lSRPyc complex on their cell surface. Upon binding IL-15P subunit activates Janus kinase 1 (Jakl) and yc subunit Janus kinase 3 (Jak3), which leads to phosphorylation and activation of signal transducer and activator of transcription 3 (STAT3) and STAT5. Due to sharing of receptor subunits between IL-2 and IL-15, both of these cytokines have similar downstream effects including the induction of Bel -2, MAP (mitogen-activated protein kinase) kinase pathway and the phosphorylation of Lek (lymphocyte-activated protein tyrosine kinase) and Syk (spleen tyrosine kinase) kinases, which leads to cell proliferation and maturation. NWVNVI SDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDAS IHDTVENLI I L ANNSLSSNGNVTESGCKECEELEEKNIKE FLQS FVHIVQMFINTS As used herein, the term “IL-15RP” has its general meaning in the art and refers to is a subunit of the interleukin 15 receptor that in humans is encoded by the IL2RB gene. The term is also known as IL-2RP and CD122. The IL-15 receptor is composed of three subunits: IL-15R alpha, CD122, and CD132. Two of these subunits, CD122 and CD132, are shared with the receptor for IL-2, but IL-2 receptor has an additional subunit (CD25). An exemplary amino acid sequence for IL-15RP is shown as SEQ ID NO:2. MAAPALSWRLPLLI LLLPLATSWASAAVNGTSQFTCFYNSRANI SCVWSQDGALQDTSCQ VHAWPDRRRWNQTCELLPVSQASWACNLI LGAPDSQKLTTVDIVTLRVLCREGVRWRVMA IQDFKPFENLRLMAPI SLQVVHVETHRCNI SWE I SQASHYFERHLE FEARTLS PGHTWEE APLLTLKQKQEWI CLETLTPDTQYE FQVRVKPLQGE FTTWS PWSQPLAFRTKPAALGKDT I PWLGHLLVGLSGAFGFI I LVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDV QKWLSS PFPSSS FS PGGLAPE I S PLEVLERDKVTQLLLQQDKVPE PASLSSNHSLTSCFT NQGYFFFHLPDALE IEACQVYFTYDPYSEEDPDEGVAGAPTGSS PQPLQPLSGEDDAYCT FPSRDDLLLFS PSLLGGPS PPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVP DLVDFQPPPELVLREAGEEVPDAGPREGVS FPWSRPPGQGE FRALNARLPLNTDAYLSLQ ELQGQDPTHLV

[0017] As used herein, the term “domain” refers to any portion of a polypeptide that adopts a tertiary structure.

[0018] The term “binding domain” as used herein refers to the one or more regions of a polypeptide that mediate specific binding with a target molecule (e.g., IL-15RP).

[0019] As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.

[0020] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0021] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0022] As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position.

[0023] As used herein, the term "conservative mutations" refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0024] As used herein, the term “IL-15 mutein” has its general meaning in the art and refers to IL-15 having at least one amino acid substitution (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitution) that replace the wild-type residue(s) .

[0025] The term “binding” as used herein refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" in the context of the binding of a polypeptide to a predetermined target molecule (e.g., receptor) typically is a binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about IO'10M or less, or about 10'11M or even less. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka(i.e., Kd / Ka) and is expressed as a molar concentration (M). Methods for measuring the KD of a polypeptide are well known in the art and include, without limitation, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the target molecule as the ligand and the polypeptide as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used. As used herein, the term “acidic pH” means a pH of 4.0 to 6.5. The term includes pH values of any one of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In particular aspects, the “acidic pH” is 5.

[0026] As used herein, the term “neutral pH” means a pH of 6.7 to about 10.0. The term includes pH values of any one of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0. In particular aspects, the “neutral pH” is 7.

[0027] As used herein, the term "subject", “host”, “individual” or “patient” refers to a mammal, preferably a human being, male or female at any age that is in-need of a therapy.

[0028] A used herein, the term "host cell" or "recipient cell" refers to a cell that was genetically engineered, i.e. harboring an exogenous nucleotide sequence, preferably stably integrated, in its genome. As used herein, the term “host immune cell” refers to a host cell that functions in an immune response or a progenitor, or progeny thereof.

[0029] As used herein, the term “engineered” refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered cell” refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the cell prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the cell prior to the manipulation; and / or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the cell prior to the manipulation. In some embodiments, a an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a polynucleotide, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. Those of ordinary skill in the art will appreciate that reference to an “engineered cell” herein may, in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such cell. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0030] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0031] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0032] As used herein, the expression "therapeutically effective amount" is meant a sufficient amount of the active ingredient of the present invention to induce an immune response at a reasonable benefit / risk ratio applicable to the medical treatment.

[0033] Muteins of the present invention:

[0034] An object of the present invention relates to an IL- 15 mutein having an amino acid sequence as set forth in SEQ ID NO: 1 comprising one or substitution(s) at position 4, 7, 10, 11, 15, 58, 62, or 72 wherein said mutein binds to IL-15RP with a higher affinity at an acidic pH than at a neutral pH.

[0035] According to the present invention the IL-5 mutein of the present invention as compared to the IL-15 WT displays pH-switchable properties. In particular, the IL-15 mutein of the present invention binds to IL-15RP with a higher affinity at a pH selected from about 4.0 to about 7.0, preferably about 5 to about 6.5, than at a pH selected from about 7 to about 7.5. More particularly, the IL- 15 mutein of the present invention does not bind to the IL-15RP at pH 7 but binds to the IL-15RP at pH 5 or lower.

[0036] Even more particularly, the IL- 15 mutein of the present invention triggers more potent STAT5 activation at acidic pH (pH 5-6.5) compared to physiological pH (pH 7). The IL-15 mutein demonstrates significantly lower EC50 values than wild-type IL- 15 under acidic conditions, indicating enhanced potency in the acidic tumor microenvironment.

[0037] Even more particularly, the IL-15 mutein of the present invention triggers more potent STAT3 and / or STAT5 activation at pH 5 than at pH 7.

[0038] Furthermore, the binding of the IL- 15 mutein to IL-15RP with a lower affinity at a pH of about 7 to about 7.5 compared to a wild-type IL-15 molecule may be characterized by a binding constant Kd which is about 0.3; about 0.5; about 0.8; about 1; about 1.5; about 2; about 2.5 or about 3 orders of magnitude higher for the IL- 15 mutein as compared for the wild-type IL- 15 molecule. The binding of the IL- 15 mutein to IL-15RP with higher affinity at a pH selected from a pH of about 4.0 to about 7.0, compared to a wild-type IL-15 molecule, may be characterized by a binding constant Kd which is about 0.3; about 0.5; about 0.8; about 1; about 1.5; about 2; about 2.5 or about 3 orders of magnitude lower for the IL-15 mutein as compared to the wild-type IL- 15 molecule.

[0039] Moreover the IL-15 mutein of the present invention may induce superior expansion of activated T cells expressing a high affinity receptor complex in an acidic micro environment (pH about 5) such as that found in the tumor micro environment (TME). Due to the higher activity of the IL-15 mutein of the present invention in the tumour micro environment (TME) and a comparably lower activity in the periphery, such as in blood, the IL-15 mutein of the present invention may overcome the problems of dose limiting toxicity which is associated with prior art IL-15 therapies. Furthermore, when used in combination with other therapeutic molecules, for example immune checkpoint inhibitors, the action of prior art IL-15 molecules limits the dose of such other molecules due to combined toxicity in the periphery. Accordingly, the selective activity of the IL- 15 mutein of the present may reduce toxicity in a combination treatment and may allow for higher doses of other therapeutic molecules, for example immune checkpoint inhibitors, and may thus increase the therapeutic effect of such treatments.

[0040] In some embodiments, the IL-15 mutein of the present invention comprises 1, 2, 3, 4, 5, 6, 7, or 8 substitution(s) at position 4, 7, 10, 11, 15, 58, 62, or 72.

[0041] In some embodiments, the IL- 15 mutein of the present invention comprises 1, 2, 3 or 4 substitution(s) at position 4, 7, 10, 11 or 15, and / or 1, 2 or 3 substitution(s) at position 58, 62 or 72.

[0042] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 4 wherein the asparagine residue (N) is substituted by an amino acid residue with a negative charged side chain. In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 4 wherein the asparagine residue (N) is substituted by an aspartic residue (D). In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 7 wherein the serine residue (S) is substituted by an asparagine residue (N).

[0043] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 10 wherein the lysine residue (K) is substituted by an arginine residue (R).

[0044] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 11 wherein the leucine lysine (K) is substituted by an amino acid residue with a hydrophobic side chain. In some embodiments, the IL-15 mutein of the present invention comprises one substitution at position 11 wherein the lysine residue (K) is substituted by a valine residue (V).

[0045] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 15 wherein the leucine residue (L) is substituted by an amino acid residue with a positive charged side chain. In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 15 wherein the leucine residue (N) is substituted by a histidine residue (H).

[0046] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 58 wherein the serine residue (S) is substituted by an amino acid residue with a hydrophobic side chain. In some embodiments, the IL-15 mutein of the present invention comprises one substitution at position 58 wherein the serine residue (S) is substituted by a tyrosine residue (Y).

[0047] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 62 wherein the serine threonine (T) is substituted by an amino acid residue with a amino acid with a hydrophobic side chain. In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 62 wherein the threonine residue (T) is substituted by a tyrosine residue (Y).

[0048] In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 72 wherein the asparagine residue (N) is substituted by an amino acid residue with a negative charged side chain. In some embodiments, the IL- 15 mutein of the present invention comprises one substitution at position 72 wherein the asparagine residue (N) is substituted by an aspartic residue (D).

[0049] In some embodiments, the IL- 15 mutein of the present invention characterised by one or more substitutions selected from the group consisting of N4D, S7N, K10R, KI IV, L15H, S58Y, T62Y and N72D.

[0050] In some embodiments, the IL-15 mutein comprises the amino acid sequence as set forth in SEQ ID NO:3.

[0051] SEQ ID NO : 3> I L- 15 mutein

[0052] NWVDVINDLRVIEDHIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVI SLESGDAYIHDYVENLI I L ANDSLSSNGNVTESGCKECEELEEKNIKEFLQS FVHIVQMFINTS

[0053] In some embodiments, the IL-15 mutein of the present invention comprises one or more conservative substitution(s).

[0054] In some embodiments, the IL-15 mutein comprises the amino acid sequence as set forth in SEQ ID NO:3 that further comprises one or more conservative substitution(s).

[0055] In some embodiments, the IL- 15 mutein of the present invention further comprises one or more heterologous sequences such as purification tags, for example: P-galactosidase, glutathione-S- transferase, green fluorescent proteins (GFP), and epitope tags such as FLAG, myc tag, poly histidine (e.g., 6HIS). In some embodiments, the IL- 15 mutein of the present invention comprises an AviTag® sequence. The AviTag® sequence (U.S. Pat. Nos. 5,932,433, 5,874,239 & 5,723,584) is a unique peptide, just 15 residues long, that is recognized by biotin ligase (Schatz P. J., 1993). In the presence of ATP, the ligase specifically attaches biotin to the lysine residue in this sequence. Using vectors, the AviTag™ can be genetically fused to a much bigger polypeptide. This feature effectively allows any polypeptide that has been cloned to be tagged with a biotin molecule. The originality of the AviTag™ is that this peptide can be biotinylated by the E. Colt enzyme BirA. Thus, polypeptides containing this biotinylated peptide either at their NH2 or at their COOH terminus can interact with very strong affinity with streptavidine and can be multimerized or attached to streptavidine-coated surfaces (Altman et al, Science, 1996; Bodinier et al, Nature Medicine, 2000; Rabu et al, J. Biol. Chem., 2005). In some embodiments, the IL- 15 mutein of the present invention further comprises the sequence of a signal peptide. As used herein, the term "signal peptide" has its general meaning in the art and refers to a pre-peptide which is present as an N-terminal peptide on a precursor form of a protein. The function of the signal peptide is to facilitate translocation of the expressed polypeptide to which it is attached into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.

[0056] Fusion nroteins and i

[0057] A further object of the present invention relates to a fusion protein that comprises the IL-15 mutein of the present invention.

[0058] As used herein, the term “fusion protein” means a protein created by joining two or more polypeptide sequences together. The fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide, e.g., an RNA-binding domain, with the nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open-reading frame. In other words, a “fusion polypeptide” or “fusion protein” is a recombinant protein of two or more proteins which are joined by a peptide bond or via several peptides. The fusion protein may also comprise a peptide linker between the two domains. Within the fusion protein, the term "operably linked" is intended to indicate that the peptide of the present invention and the heterologous polypeptide are fused in-frame to each other.

[0059] In some embodiments, the fusion protein comprises the IL- 15 mutein of the present invention that is fused to one immunoglobulin domain.

[0060] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. In typical IgG antibodies, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate in the antibody binding site, or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (Kabat et al., 1992, hereafter “Kabat et al ”). The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31- 35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. For the agonist antibodies described hereafter, the CDRs have been determined using CDR finding algorithms from www.bioinf.org.uk - see the section entitled « How to identify the CDRs by looking at a sequence » within the Antibodies pages.

[0061] As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (such as e.g., a chain of a conventional 4-chain antibody or of a heavy chain antibody or light chain), or to a polypeptide that essentially consists of such a globular region.

[0062] In some embodiments, the fusion protein comprises the IL- 15 mutein of the present invention that is fused to a heavy chain of an antibody.

[0063] In some embodiments, the heavy chain is fused to the IL- 15 fusion mutein via a linker.

[0064] As used herein, the term “linker” has its general meaning in the art and refers to an amino acid sequence of a length sufficient to ensure that the proteins form proper secondary and tertiary structures. Typically, linkers are those which allow the compound to adopt a proper conformation. The most suitable linker sequences (1) will adopt a flexible extended conformation, (2) will not exhibit a propensity for developing ordered secondary structure which could interact with the functional domains of fusion proteins, and (3) will have minimal hydrophobic or charged character which could promote interaction with the functional protein domains.

[0065] Typically linkers can vary from 10 to 30 amino acids in length. In some embodiments, the linker is a Gly / Ser linker and comprises one or more repeats of the amino acid sequence GGGS (SEQ ID NO:4) or GGGGS (SEQ ID NO:5)

[0066] In some embodiments, the heavy chain comes from an antibody having specificity for a checkpoint molecule.

[0067] As used herein, the term “checkpoint molecule” refers to a negative regulators of immune responses, such as co-stimulatory receptors occurring on the surface of several immune cells and ligands to said receptors. The checkpoint molecule may be selected from CD27, CD 137, 2B4, TIGIT, CD155, CD160, ICOS, HVEM, CD40L, LIGHT, LAIR1, 0X40, DNAM-1, PD- Ll, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7- H3, B7-H4, BTLA, IDO1, ID02, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0068] In some embodiments, the heavy chain comes from an antibody having specificity of PD-1. As used herein, the term “PD-1” has its general meaning in the art and refers to programmed cell death protein 1 (also known as CD279). PD-1 acts as an immune checkpoint, which upon binding of one of its ligands, PD-L1 or PD-L2, enables Shp2 to dephosphorylate CD28 and inhibits the activation of T cells.

[0069] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX- 1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg).

[0070] In some embodiments, the anti-PD-1 antibody is the anti-PDIGepi 135c as disclosed in W02016020856 and in Fenwick, Craig, et al. "Tumor suppression of novel anti-PD-1 antibodies mediated through CD28 costimulatory pathway." Journal of Experimental Medicine (2019): jem-20182359.

[0071] In some embodiments, the heavy chain of the present invention comprises the VH domain as set forth in SEQ ID NO:6, or 7.

[0072] SEQ ID NO : 6 > VH domain of pembroli zumab

[0073] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVT LTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

[0074] SEQ ID NO : 7 > VH domain of nivolumab QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFT ISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

[0075] In some embodiments, the heavy chain comprises an IgG Fc region of an IgG4 immunoglobulin.

[0076] In some embodiments, the heavy chain consists of the amino acid sequence as set forth in SEQ ID NO:8 or 9. SEQ ID NO : 8 > heavy chain o f pembroli zumab QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFT I SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTI SKAKGQPRE PQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK

[0077] SEQ ID NO : 9 > heavy chain o f nivolumab QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVT LTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTI SKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0078] In some embodiments, the heavy chain comes from an antibody having specificity for a tumor antigen that is typically selected from the group consisting of EpCAM, EGFR, HER-2, HER- 3, c-Met, FoIR, PSMA, CD38, BCMA, CEA, 5T4, AFP, B7- H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD40, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, CLDN18.2, DLL3, EphA2, ED-B fibronectin, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Mucl, Mucl6, NaPi2b, Nectin-4, P-cadherin, NY-ESO- 1, PRLR, PSCA, PTK7, R0R1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, or WT1. A polypeptide binding domain may bind a haematologic tumor antigen; a haematologic tumor antigen may be expressed by lymphoid cells. Tumour antigens of this type may include, for example, ADIR, AURKA, BCR-ABL, BMI1, CML28, CML66, Cyclin Al, DDX3Y, DKK1, FMOD, FRAME, G250 / CAIX, HAGE, HM1.24, hTERT, LPP, MAG EA3, MAGEA3, MEF2D, MLL, MPP1, MUC1, Myeloperoxidase, NEWREN60, NY-ESO- 1, PANE1, PRAME, Proteinase 3, PTPN20A / B, RHAMM, R0R1, SLAMF7, Survivin, TEX14, WT1, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD52, CD123, CD269, CD138, HM1 .24, SLAMF7. The term (haematologic) tumour antigen may include, for example, surface antigens such as CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD52, CD123, CD269, CD 138, HM1.24, SLAMF7.

[0079] A further object of the present invention relates to an immunocytokine that comprises a heavy chain of an antibody that is fused to the IL-15 fusion mutein of the present invention. As used herein, the term “immunocytokine” refers to an antibody directly or indirectly linked by covalence to a cytokine or derivates thereof. Said antibody and said cytokine can be linked by a linker.

[0080] In some embodiments, the immunocytokine of the present invention has specificity for a checkpoint molecule. In some embodiments, the immunocytokine of the present invention has specificity for PD-1. In some embodiments, the immunocytokine of the present invention has specificity for a tumor antigen. Thus in some embodiments, the immunocytokine of the present invention comprises a heavy chain as described above.

[0081] In some embodiments, the immunocytokine of the present invention comprises a heavy chain that consists of the amino acid sequence as set forth in SEQ ID NO:6 or 7.

[0082] In some embodiments, the immunocytokine of the present invention comprises a heavy chain a set forth in SEQ ID NO:6 and a light chain a set forth in SEQ ID NO: 10.

[0083] In some embodiments, the immunocytokine of the present invention comprises a heavy chain a set forth in SEQ ID NO: 7 and a light chain a set forth in SEQ ID NO: 11.

[0084] SEQ ID NO : 10 > light chain o f pembroli zumab

[0085] E IVLTQS PATLSLS PGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGS GSGTDFTLTI SSLE PEDFAVYYCQHSRDLPLTFGGGTKVE IKRTVAAPSVFI FPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKS FNRGEC

[0086] SEQ ID NO : 11> light chain o f nivolumab

[0087] E IVLTQS PATLSLS PGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGT DFTLTI SSLE PEDFAVYYCQQSSNWPRTFGQGTKVE IKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTK S FNRGEC

[0088] Polynucleotides, vectors and host cells of the present invention:

[0089] A further object of the invention relates to a polynucleotide that encodes for the IL- 15 mutein of the present invention.

[0090] A further object of the invention relates to a polynucleotide that encodes for the fusion protein of the present invention. A further object of the invention relates to a polynucleotide that encodes for the immunocytokine of the present invention.

[0091] Typically, said polynucleotide is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0092] So, a further object of the invention relates to a vector comprising a polynucleotide of the present invention.

[0093] Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of 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 human antibody C region can be inserted and expressed. Examples of 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. Other examples of 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 replicationdefective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0094] A further object of the present invention relates to a host cell which has been transfected, infected or transformed by a polynucleotide and / or a vector according to the present invention.

[0095] The polynucleotides of the invention may be used to produce the IL- 15 mutein of the present invention in a suitable expression system. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts. Mammalian host cells include Chinese Hamster Ovary (CHO cells) including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker, CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, for example GS CHO cell lines together with GS Xceed™ gene expression system (Lonza), or HEK cells.

[0096] The present invention also relates to a method of producing a recombinant host cell expressing the IL-15 mutein of the present invention, said method comprising the steps of (i) introducing in vitro or ex vivo a recombinant polynucleotide or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said fusion protein.

[0097] The host cell as disclosed herein are thus particularly suitable for producing the IL-15 mutein of the present invention. Indeed, when recombinant expression are introduced into mammalian host cells, the polypeptides are produced by culturing the host cells for a period of time sufficient in the host cells and, optionally, secreted into the culture medium in which the host cells are grown. The polypeptides can be recovered and purified for example from the culture medium after their secretion using standard protein purification methods.

[0098] In some embodiments, the host cell is a host immune cell that can be subsequently used for therapeutic purposes. Thus, in some embodiments, the host immune cell is thus engineered to express the IL-15 mutein of the present invention.

[0099] In some embodiments, the host immune cell is selected from the group consisting of monocytes, macrophages, NK cells, B cells, MAIT cells, and T cells.

[0100] In some embodiments, the host immune cell is a NK cell.

[0101] As used herein, the term “NK cell” has its general meaning in the art and refers to a subpopulation of lymphocytes that is involved in non-conventional immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and / or NKp46 for human NK cells, the absence of the alpha / beta or gamma / delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express “self’ MHC / HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells.

[0102] In some embodiments, the host immune cell of the present invention expresses an endogenous TCR.

[0103] As used herein, the term “TCR” has its general meaning in the art and refers to the molecule found on the surface of T cells that is responsible for recognizing antigens bound to MHC molecules. The TCR heterodimer consists of an alpha and beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains. Engagement of the TCR with antigen and MHC results in activation of its T lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N- terminal “immunoglobulin (Ig)-variable (V) domain”, one “Ig-constant (C) domain”, a “transmembrane region”, and a short “cytoplasmic tail” at the C-terminal end. The constant domain of the TCR consists of short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. The structure allows the TCR to associate with other molecules like CD3 which possess three distinct chains (y, 5, and a) in mammals and the ^-chain. These accessory molecules have negatively charged transmembrane regions and are vital to propagating the signal from the TCR into the cell. The CD3 chains, together with the TCR, form what is known as the TCR complex. The signal from the TCR complex is enhanced by simultaneous binding of the MHC molecules by a specific co-receptor. On helper T cells, this co-receptor is CD4 (specific for class II MHC); whereas on cytotoxic T cells, this co-receptor is CD8 (specific for class I MHC). The co-receptor not only ensures the specificity of the TCR for an antigen, but also allows prolonged engagement between the antigen presenting cell and the T cell and recruits essential molecules (e.g., LCK) inside the cell involved in the signaling of the activated T lymphocyte.

[0104] In some embodiments, the host immune cell of the present invention expresses an exogenous TCR (i.e. the host cell is engineered for expressing a TCR of interest) as described in Schober, Kilian, et al. "Orthotopic replacement of T-cell receptor a-and fl-chains with preservation of near-physiological T-cell function. " Nature biomedical engineering 3.12 (2019): 974-984 and Rohaan, M. W., et al. "MART-1 TCR gene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I / IIa clinical trial. " Immuno-Oncology and Technology 15 (2022): 100089.

[0105] As used herein, the term “exogenous T cell receptor” or “exogenous TCR” refers to a recombinant TCR which is expressed in a host immune cell by introduction of exogenous polynucleotides encoding for a TCR, i.e. one polynucleotide encoding for the alpha chain and one polynucleotide encoding for the beta chain. In particular, the exogenous TCR may be expressed in a cell in which the TCR is either not natively expressed or is expressed at levels that are insufficient to induce a response by the cell or a responder cell upon TCR ligand binding.

[0106] In some embodiments, the host immune cell is a hematopoietic cell from the lymphoid lineage that comprises CD4+ and CD8+ T cells. Non-limiting examples of host immune cells of the lymphoid lineage include T cells, and precursors thereof including embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells may be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), and y5 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

[0107] In some embodiments, the host immune cell is a T cell.

[0108] As used herein, the term “T cell” has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity. T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility. There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, and gamma delta T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

[0109] The T cell can be a CD4+ T cell or a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell.

[0110] In some embodiments, the host immune cell of the present invention is engineered to express the IL- 15 mutein of the present invention and a CAR.

[0111] As used herein, the term “chimeric antigen receptor” or “CAR” has its general meaning in the art and comprises one or more artificially constructed hybrid polypeptides containing an antigen binding domain linked to one or more T- cell signalling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting e.g. the antigen-binding properties of monoclonal antibodies. The chimeric antigen receptor of the present invention typically comprises one more polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain.

[0112] In some embodiments, the host immune cell is a CAR-T cell.

[0113] As used herein the term "CAR-T cell" refers to a T lymphocyte that has been genetically engineered to express a CAR. The T lymphocytes that are genetically modified may be "derived" or "obtained" from the patient who will receive the treatment using the genetically modified T cells or they may be "derived" or "obtained" from a different patient.

[0114] In some embodiments, the host immune cell is a pluripotent stem cell (PSC). PSCs can be indeed be modified by a CAR and then can be used for deriving T cells (e.g. WO 2017100403). PSCs include embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs). iPSCs can be generated directly from adult cells (e.g., somatic cells). iPSCs can be typically derived or generated by introducing a specific set of pluripotency-associated genes, or "reprogramming factors", into a given cell type. Reprogramming factors include, but are not limited to, OCT4 (also known as "POU5FL"), SOX2, cMYC, and KLF4, which are also known as Yamanaka factors. See Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors". Cell 126 (4): 663-76. The CAR of the invention may be a first generation, a second generation, or a third generation CAR as described hereabove. Preferably, the CAR is a second or third generation CAR. Typically, “first-generation CARs” contain a single signalling domain. CARs containing a signalling domain together with one additional costimulatory domain are termed “second generation” while those containing a signalling domain together with two additional costimulatory domains are listed as “third generation”. For example, first-generation CARs contain solely the CD3(^ chain as a single signalling domain. Second- and third-generation CARs consist of one or two additional costimulatory signalling domains, respectively, such as CD28, CD27, OX-40 (CD134) and 4-1BB (CD137). For example, second-generation CAR may contain CD3(^ and CD28 signalling domains, while third-generation CAR may contain CD3(^, CD28 and either 0X40 (CD134) or 4-1BB (CD137). “TRUCKs” represent the recently developed “fourth-generation” CARs. TRUCKs (T cells redirected for universal cytokine killing) are CAR-redirected T cells used as vehicles to produce and release a transgenic product that accumulates in the targeted tissue. The product, for example a pro-inflammatory cytokine, may be constitutively produced or induced once the T cell is activated by the CAR. Other substances such as enzymes or immunomodulatory molecules may be produced in the same way and deposited by CAR-redirected T cells in the targeted lesion. This strategy involves two separate transgenes expressing for example (i) the CAR and (ii) a cell activation responsive promoter linked to a cytokine such as IL-12. Consequently, immune stimulatory cytokine such as IL-12 is secreted upon CAR engagement. In a particular embodiment, the CAR is a CAR of fourth generation as defined above.

[0115] In some embodiments, the CAR of the present invention consists in a TCR mimic. TCR mimics were recently developed in which the Va and Vb domains of native TCR were replaced by the VH and VL domains of an antibody specific for a given antigen. They confer HLA-independent recognition of antigen and coincidently permit to benefit of the high antigen sensitivity supplied by the structure of the native TCR-CD3 architecture. They are denoted as “Synthetic TCR and Antigen Receptor” or “STAR” as described in / . / > / , Yue, etal. "Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors. " Science Translational Medicine 13.586 (2021): eabb5191.,' Wang, Jiasheng, et al. "A Novel Adoptive Synthetic TCR and Antigen Receptor (STAR) T - Cell Therapy for B - Cell Acute Lymphoblastic Leukemia. " American Journal of Hematology (2022) and in W02020029774 that are incorporated by reference or “HLA-independent T cell receptor” or “HIT receptor” as described mMansilla- Soto, J., Eyquem, J., Haubner, S. et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 28, 345- -352 (2022), and in WO2019157454 that are incorporated by reference. Upon expression in human T cells, STARs and HIT receptors mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. The STAR and HIT architecture cannot incorporate a CD28 transmembrane domain. Moreover, steric hindrance limits the benefits expected from the incorporation of the CD28 intracytoplasmic segment at the carboxy-terminus of the TCR a or b chains (Wang, Jiasheng, et al. "A Novel Adoptive Synthetic TCR and Antigen Receptor (STAR) T - Cell Therapy for B - Cell Acute Lymphoblastic Leukemia. " American Journal of Hematology (2022)). Therefore, host immunes cells armed with STAR and HIT receptors will be particularly prone to benefit of the costimulatory signals provided by co-expressed the Carmil2 polypeptide of the present invention.

[0116] In some embodiments, the CAR of the present invention thus derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domains are substituted by a variable domain of an antibody.

[0117] In some embodiments, the CAR of the present invention derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a first variable domain of an antibody (e.g. a VL or VH domain) and the immunoglobulin Invariable (V) domain of the beta chain is substituted by a second variable domain of an antibody (e.g. a VL or VH domain) wherein the first variable domain is capable of dimerizing with the second variable domain to form a fragment variable (Fv) that binds to the antigen of interest.

[0118] In some embodiments, the CAR of the present invention derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a VL domain of an antibody and the immunoglobulin (Ig)-variable (V) domain of the beta chain is substituted by a VH domain of an antibody wherein the VL domain is capable of dimerizing with the VH domain to form a fragment variable (Fv) that binds to the antigen of interest.

[0119] According to the present embodiments, the CAR is thus capable of associating with a CD3 complex to form the T-cell co-receptor. In some embodiments, the CD3 complex comprises a CD3y chain, a CD35 chain, and two CD3s chains. In some embodiments, the CAR of the present invention and the CD3 complex form an antigen recognizing receptor complex similar to a native TCR / CD3 complex. In some embodiments, the CAR of the present invention replaces a native and / or an endogenous TCR in the CD3 / TCR complex of host immune cell.

[0120] In some embodiments, the host immune cell expresses a TCR or CAR that is specific for a tumor antigen. Non-limiting examples of tumor antigens include carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (LI CAM), melanoma antigen family A, 1 (MAGE- Al), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME and ERBB.

[0121] Methods

[0122] A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the IL-15 mutein of the present invention.

[0123] A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotide of the present invention. A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the fusion protein of the present invention.

[0124] A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the immunocytokine of the present invention.

[0125] A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of host immune cells of the present invention.

[0126] In particular, the methods of the present invention is particularly suitable for the treatment of cancer.

[0127] As used herein, the term "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 diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; 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; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0128] The IL-15 mutein of the present invention is particularly suitable for treating muscle cachexia and muscle wasting disorders. The pH-dependent properties allow for enhanced activity in the acidic microenvironment often present in diseased muscle tissue, while reducing systemic exposure and associated side effects.

[0129] In particular, the IL-15 fusion mutein of the present invention is particularly suitable for enhancing the proliferation, migration, persistence and / or activity of T cells in a subject in need thereof. In particular, the IL-15 fusion mutein of the present invention is particularly suitable for enhancing the proliferation, migration, persistence and / or activity of T CD4+cells. In particular, the IL- 15 fusion mutein of the present invention is particularly suitable for enhancing the proliferation, migration, persistence and / or activity of T CD8+cells. In particular, the IL-15 fusion mutein of the present invention is particularly suitable for enhancing the proliferation, migration, persistence and / or activity of Gamma delta T cells. In particular, the IL- 15 fusion mutein of the present invention is particularly suitable for enhancing the proliferation, migration, persistence and / or activity of CAR-T cells.

[0130] Thus a further object of the present invention provides a method of therapy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the IL- 15 mutein of the present invention , wherein said administration enhances the proliferation, migration, persistence and / or activity of T cells in the subject.

[0131] More particularly, the present invention provides a method of reducing T cell exhaustion in a subject in need thereof comprising administering to the subject a therapeutically effective amount the IL- 15 mutein of the present invention.

[0132] In some embodiments, the IL-15 mutein of the present invention is administered in combination with chemotherapy.

[0133] As used herein, the term “chemotherapy” has its general meaning in the art and refers to the treatment that consists in administering to the patient a chemotherapeutic agent. As used herein, the term “chemotherapeutic agent” refers to a chemical compound that is (i.e., drug) or becomes (i.e., prodrug), for example, selectively destructive or selectively toxic to malignant cells and tissues, xamples of chemotherapeutic agents include but not limited to: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; acetogenins, e.g., bullatacin and bullatacinone; a camptothecin, including synthetic analog topotecan; bryostatin, callystatin; CC-1065, including its adozelesin, carzelesin, and bizelesin synthetic analogs; cryptophycins, particularly cryptophycin 1 and cryptophycin 8; dolastatin; duocarmycin, including the synthetic analogs KW-2189 and CBI- TMI; eleutherobin; 5 -azacytidine; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, glufosfamide, evofosfamide, bendamustine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin phill), dynemicin including dynemicin A, bisphosphonates such as clodronate, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores, aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogs such as demopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as cladribine, pentostatin, fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replinishers such as frolinic acid; radiotherapeutic agents such as Radium-223, 177-Lu-PSMA-617; trichothecenes, especially T-2 toxin, verracurin A, roridin A, and anguidine; taxoids such as paclitaxel (TAXOL®), abraxane, docetaxel (TAXOTERE®), cabazitaxel, BIND-014, tesetaxel; platinum analogs such as cisplatin and carboplatin, NC-6004 nanoplatin; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; phenamet; pirarubicin; losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid; 2- ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; trabectedin, triaziquone; 2,2Z,2ZZ-trichlorotriemylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ); cyclophosphamide; thiopeta; chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitroxantrone; vancristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; NUC-1031; FOLFOX (folinic acid, 5-fluorouracil, oxaliplatin); FOLFIRI (folinic acid, 5-fluorouracil, irinotecan); FOLFOXIRI (folinic acid, 5-fluorouracil, oxaliplatin, irinotecan), FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, oxaliplatin), and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Such agents can be conjugated onto an antibody or any targeting agent described herein to create an antibody-drug conjugate (ADC) or targeted drug conjugate.

[0134] In some embodiments, the IL-15 mutein of the present invention is administered in combination with targeted therapy.

[0135] As used herein, the term “targeted therapy” refers to a therapy targeting a particular class of proteins involved in tumor development or oncogenic signaling. In particular, targeted therapy includes administration of tyrosine kinase inhibitors (TKIs). The term “tyrosine kinase inhibitor” (“TKI”) as used herein broadly refers to agents or compounds which are capable of selectively inhibiting tyrosine kinases family of enzymes but do no not target serine or threonine kinases, including those that inhibit MAPK (mitogen-activated protein kinases). The TKI may inhibit tyrosine kinase activity by directly acting on a tyrosine kinase molecule, or it may cooperate with one or more other factors or agents to achieve the desired inhibition. The tyrosine kinase family of enzymes includes both receptor tyrosine kinases and non-receptor tyrosine kinases. For instance, TKIs may target epidermal growth factor receptors (EGFRs) and receptors for fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). Examples of TKIs include, but are not limited to sorafenib, tepotinib, cabozantinib, tivantinib, lenvatinib, afatinib, ARQ-087, asp5878, AZD3759, AZD4547, bosutinib, brigatinib, cediranib, crenolanib, dacomitinib, dasatinib, dovitinib, E-6201, erdafitinib, erlotinib, gefitinib, gilteritinib (ASP-2215), FP-1039, HM61713, icotinib, imatinib, KX2-391 (Src), lapatinib, lestaurtinib, midostaurin, nintedanib, ODM-203, osimertinib (AZD-9291), ponatinib, poziotinib, quizartinib, radotinib, rociletinib, sulfatinib (HMPL-012), sunitinib, and TH-4000. In some embodiments, the targeted therapy comprises at least one anti-VEGF agent. As used herein an "anti- VEGF agent" refers to a molecule that inhibits Vascular endothelial growth factor (VEGF) -mediated angiogenesis. For example, an anti-VEGF therapeutic may be an antibody or a Approved anti-VEGF agents useful in the present invention include but are not limited to bevacizumab (Avastin®, Genentech / Roche) an anti-VEGF monoclonal antibody; ramucirumab (Cyramza®, Eli Lilly), an anti-VEGFR-2 antibody and ziv-aflibercept, also known as VEGF Trap (Zaltrap®; Regeneron / Sanofi). VEGFR inhibitors, such as regorafenib (Stivarga®, Bayer); vandetanib (Caprelsa®, AstraZeneca); axitinib (Inlyta®, Pfizer); and lenvatinib (Lenvima®, Eisai); Raf inhibitors, such as sorafenib (Nexavar®, Bayer AG and Onyx); dabrafenib (Tafinlar®, Novartis); and vemurafenib (Zelboraf®, Genentech / Roche); MEK inhibitors, such as cobimetanib (Cotellic®, Exelexis / Genentech / Roche); trametinib (Mekinist®, Novartis); Bcr-Abl tyrosine kinase inhibitors, such as imatinib (Gleevec®, Novartis); nilotinib (Tasigna®, Novartis); dasatinib (Spry cel®, BristolMyersSquibb); bosutinib (Bosulif®, Pfizer); and ponatinib (Inclusig®, Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such as gefitinib (Iressa®, AstraZeneca); erlotinib (Tarceeva®, Genentech / Roche / Astellas); lapatinib (Tykerb®, Novartis); afatinib (Gilotrif®, Boehringer Ingelheim); osimertinib (targeting activated EGFR, Tagrisso®, AstraZeneca); and brigatinib (Alunbrig®, Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, such as cabozanitib (Cometriq®, Exelexis); and multikinase inhibitors, such as sunitinib (Sutent®, Pfizer); pazopanib (Votrient®, Novartis); and ALK inhibitors, such as crizotinib (Xalkori®, Pfizer).

[0136] In some embodiments, the IL-15 mutein of the present invention is administered in combination with immunotherapy. As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agenti.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. In some embodiments, the immunotherapy consists in administering the patient with at least one immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune checkpoint molecule.

[0137] A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM- 3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist.

[0138] Examples of immune checkpoint inhibitors that can be used in the preoperative adjuvant immunotherapy include anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti -TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

[0139] Examples of PD-1 and PD-Ll antibodies are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO201 1082400, and WO2011161699. In some embodiments, the anti-PDl antibody is selected from the group consisting of Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo) and Camrelizumab and Tislelizumab. In some embodiments, the anti- PD-L1 antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, LY3300054, BGB-A333, SHR-1316, CK-301, and combinations thereof.

[0140] Pharmaceutical compositions:

[0141] A further object of the present invention relates to a pharmaceutical composition comprising the IL- 15 mutein of the present invention and a pharmaceutically acceptable carrier. A further object of the present invention relates to a pharmaceutical composition comprising the polynucleotide of the present invention and a pharmaceutically acceptable carrier.

[0142] A further object of the present invention relates to a pharmaceutical composition comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier.

[0143] A further object of the present invention relates to a pharmaceutical composition comprising the immunocytokine of the present invention and a pharmaceutically acceptable carrier.

[0144] A further object of the present invention relates to a pharmaceutical composition comprising the population of host immune cells of the present invention and a pharmaceutically acceptable carrier.

[0145] Pharmaceutically acceptable carriers that may be used in these compositions 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, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions 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, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, 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 polyoxyethylated versions. 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. Other commonly used 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 compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, polypeptide of the present invention in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for a polypeptide of the present inventon in a pharmaceutical composition of this invention may between about 1 mg / m2and 500 mg / m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular polypeptide in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g., 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g., about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the polypeptide of the invention.

[0146] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0147] FIGURES:

[0148] Figure 1: Switchkine direct evolution based on yeast display. The schematic graph represents the IL-15 library expressed at the yeast surface with in red the mutated amino acids (left). The library is screened using biotinylated IL-15Ra or IL-15R0 ectodomain at pH 5 led to the identification of the different mutants. Histogram overlays displaying IL-15Ra or IL- 15R0 staining of the library at each round of selection at pH 5 are shown (right). Figure 2: Switch-150 yeast binding assay. Dose-dependent binding at pH7 and pH5 of IL- 15R0 serial dilutions to IL-15 and Switch-150 displaying yeasts.

[0149] Figure 3: pH-Dependent Signaling of IL-15 WT and IL-15 mutein 0 in YT Cells. The signaling of IL-15 mutein 0 was assessed by measuring pSTAT5 levels at physiological (pH 7.5) and acidic (pH 6.5) pH in YT cell line. The table shows the EC50 values (nM) for IL-15 WT and IL- 15 mutein 0 at each pH.

[0150] EXAMPLE:

[0151] Methods

[0152] Protein production

[0153] Human IL-15Ra ectodomain and IL-15R0 were cloned in the pFB-CTlOHF vector in frame with the N-terminal gp67, with a C-terminal biotin acceptor peptide (BAP)- LNDIFEAQKIEWHW followed by a histidine tag. Proteins were produced using the baculovirus expression system. Briefly, vectors were recombined in DHIOBac bacteria (Gibco), and the generated bacmid was used to generate the baculovirus. Baculovirus was produced and amplified in Sf9 cells and used to infect High Five cells for protein expression. Two days after infection, His-Pur Ni-nitrilotriacetic acid (NTA) resin (Invitrogen, 88222) was used to capture the proteins released in the cell culture supernatant. Proteins were purified by size exclusion chromatography on a Superdex 75 Increase column (GE Healthcare, 29-1487-21). Proteins were conserved in 10 mM Hepes (pH 7.2) and 150 mM NaCl (Hepes-buffered saline). The proteins were reduced with 10 mM cysteine, alkylated with 20 mM iodoacetamide, and biotinylated with BirA ligase in the presence of 100 pM biotin.

[0154] Generation and selection of IL-15 library

[0155] Adapting a previously described protocol for yeast display, we cloned IL-15 cDNA in the pCT302 vector for the expression in yeast. The IL-15 library was generated assembling eight overlapping primers, among which two of them contained the homology regions necessary for the combination with the pCT302 vector). Three of the primers had NDT codons (encoding for Gly, Vai, Leu, He, Cys, Ser, Arg, His, Asp, Asn, Phe, and Tyr amino acids) used to randomly mutate different residues at the interfaces with the receptors. The polymerase chain reaction (PCR) product was further amplified using Lib Fw and Lib Rv primers, at a final concentration of 10 pM, to obtain at least 25 pg of DNA.

[0156] Saccharomyces cerevisiae strain EBY100 was transformed by electroporation with 25 pg of insert DNA and 5 pg of the linearized and purified plasmid. Transfected yeasts were grown in synthetic defined medium with casamino acids (SDCAA) medium for 1 day at 30°C and in synthetic defined medium with casamino acids and galactose (SGCAA) for 2 days at 20°C at each round of selection. The library, with a size of 2 * 108, was screened by MACS using an LS column (Miltenyi, 130-042-401): The first round of selection was carried out with 1010cells, and the subsequent ones were carried out with 108cells to ensure at least 10-fold coverage for each round. Biotinylated IL-15Ra and Rb ectodomain were used at different concentrations to select pH-resistant IL-15 variants: The first two rounds were performed using IL-15Ra or Rb tetramer at 100 nM in pH 5, and the third and fourth rounds were performed with 1 pM and 100 nM IL-15Ra or Rb monomer, respectively. IL-15Ra and Rb tetramers were generated by incubating IL-2Ra and streptavidin (SA)-AF647 at a ratio of 4: 1.

[0157] Results

[0158] To obtain pH-switchable IL-15 mutants, we used yeast surface display to generate large IL-15 mutant library (108variants) with varying degree of affinity towards pH-sensitive receptor subunit and engineer desirable variants using direct evolution approaches based on yeast display platform. Based on the wild-type (WT) IL-15 / IL-15R complex structure, we mutated selected amino acid residues at the interface with the IL-15R subunit of interest and clone the library of IL-15 variants in a vector for expression in yeast. The direct evolution of IL-15 is based on displaying the IL-15 library on the surface of the yeast and to evaluate the ability of displayed IL-15 variants to bind to the biotinylated IL-15R ectodomain which is used as a FACS staining and sorting reagent in combination with fluorescent streptavidin. Individual colonies from each round have been picked, sequenced, and characterized for IL-15R binding at pH 7.5 and 6.5 using the cytokine receptor subunit of interest.

[0159] The screening for IL- 15 mutants with pH-dependent binding for IL-15R0 led to the identification of the Switch- 150 presented mutations for amino acids N4D, S7N, KI OR, KI IV, L15H, S58Y, T62Y and N72D at the interface with the IL-15R0 receptor. The binding of IL- 15 to IL-15R0 is completely disrupted at low pH. The Switch-150 mutant displayed stronger binding at low pH as compared to the IL- 15 WT. However, the mutant strongly differed for their ability of binding to IL- 15Rb at physiological pH. Indeed, Switch- 15b did not interact with the IL-15Rb receptor at pH 7, thus displaying promising pH switchable properties (Figure 2).

[0160] The signaling of IL-15 mutein P was assessed by measuring pSTAT5 levels at physiological (pH 7.5) and acidic (pH 6.5) pH in YT cell line. IL-15 mutein P showed comparable signaling to IL- 15 WT at pH 7.5, with increased signaling observed at pH 6.5. Then, the IL- 15 mutein P demonstrating a lower EC50 compared to IL- 15 WT, indicating higher potency at both pH conditions (Figure 3). Our detailed mutagenesis and signaling analysis have confirmed that this mutant exhibit pH-resistant rather than pH-switchable properties. While the variant contains the established N72D mutation known for high-affinity IL-15RP interaction, our additional engineered mutations include a critical histidine residue (L15H) whose presence is essential for maintaining pH-resistant signaling properties. Comparative analysis with and without the N72D mutation demonstrates that the complete mutein preserves enhanced activity in acidic pH conditions (data not shown).

[0161] REFERENCES:

[0162] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0163] 1. Ozaki, K. & Leonard, W. J. Cytokine and Cytokine Receptor Pleiotropy and Redundancy *. Journal of Biological Chemistry 277, 29355-29358 (2002).

[0164] 2. Mitra, S. & Leonard, W. J. Biology of IL-2 and its therapeutic modulation: Mechanisms and strategies. J. Leukoc. Biol. 103, 643-655 (2018).

[0165] 3. Conlon, K. C. et al. Redistribution, Hyperproliferation, Activation of Natural Killer Cells and CD8 T Cells, and Cytokine Production During First-in-Human Clinical Trial of Recombinant Human Interleukin- 15 in Patients With Cancer. J Clin Oncol 33, 74-82 (2015).

[0166] 4. Waldmann, T. A. et al. Safety (toxicity), pharmacokinetics, immunogenicity, and impact on elements of the normal immune system of recombinant human IL- 15 in rhesus macaques. Blood 117, 4787-4795 (2011).

[0167] 5. Gorby, C., Martinez-Fabregas, J., Wilmes, S. & Moraga, I. Mapping Determinants of Cytokine Signaling via Protein Engineering. Frontiers in Immunology 9, (2018).

[0168] 6. Boedtkjer, E. & Pedersen, S. F. The Acidic Tumor Microenvironment as a Driver of Cancer. Annu Rev Physiol 82, 103-126 (2020). 7. Burkholder, B. et al. Tumor-induced perturbations of cytokines and immune cell networks. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1845, 182-201 (2014).

[0169] 8. Huber, V. et al. Cancer acidity: An ultimate frontier of tumor immune escape and a novel target of immunomodulation. Seminars in Cancer Biology 43, 74-89 (2017). 9. Wu, H. et al. T-cells produce acidic niches in lymph nodes to suppress their own effector functions. Nature Communications 11, 4113 (2020).

[0170] 10. Gaggero, S. et al. IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein. Science Immunology 7, eade5686 (2022).

Claims

CLAIMS:

1. An IL- 15 mutein having an amino acid sequence as set forth in SEQ ID NO: 1 comprising one or substitution(s) at position 4, 7, 10, 11, 15, 58, 62, or 72 wherein said mutein binds to IL-15RP with a higher affinity at an acidic pH than at a neutral pH.

2. The IL-15 mutein according to claim 1 that comprises 1, 2, 3, 4, 5, 6, 7, or 8 substitution(s) at position 4, 7, 10, 11, 15, 58, 62, or 72.

3. The IL- 15 mutein according to claim 2 that comprises 1, 2, 3 or 4 substitution(s) at position 4, 7, 10, 11 or 15, and / or 1, 2 or 3 substitution(s) at position 58, 62 or 72.

4. The IL-15 mutein according to any one of claims 1 to 3 that comprises; one substitution at position 4 wherein the asparagine residue (N) is substituted by an amino acid residue with a negative charged side chain, preferably by an aspartic residue (D), and / or, one substitution at position 7 wherein the serine residue (S) is substituted by an asparagine residue (N), and / or, one substitution at position 10 wherein the lysine residue (K) is substituted by an arginine residue (R), and / or, one substitution at position 11 wherein the leucine lysine (K) is substituted by an amino acid residue with a hydrophobic side chain, preferably by a valine residue (V), and / or, one substitution at position 15 wherein the leucine residue (L) is substituted by an amino acid residue with a positive charged side chain, preferably by a histidine residue (H), and / or, one substitution at position 58 wherein the serine residue (S) is substituted by an amino acid residue with a hydrophobic side chain, preferably by a tyrosine residue (Y), and / or,one substitution at position 62 wherein the serine threonine (T) is substituted by an amino acid residue with an amino acid with a hydrophobic side chain, preferably by a tyrosine residue (Y), and / or, one substitution at position 72 wherein the asparagine residue (N) is substituted by an amino acid residue with a negative charged side chain, preferably by an aspartic residue (D).

5. The IL-15 mutein according to any one of claims 1 to 4 that is characterized by one or more substitutions selected from the group consisting of N4D, S7N, K10R, KI IV, L15H, S58Y, T62Y and N72D.

6. The IL- 15 mutein according to any one of claims 1 to 5 that comprises the amino acid sequence as set forth in SEQ ID NO:3.

7. A fusion protein comprising the IL- 15 mutein according to any one of claims 1 to 6.

8. An immunocytokine that comprises a heavy chain of an antibody that is fused to the IL- 15 fusion mutein according to any one of claims 1 to 6.

9. The immunocytokine according to claim 8 that has specificity for a checkpoint molecule, preferably for PD-1.

10. A polynucleotide that encodes for- the IL- 15 mutein according to any one of claims 1 to 6, or- the fusion protein according to claim 7, or the immunocytokine according to claim 8 or 9.I L A vector that comprises the polynucleotide of claim 10.

12. A host cell that has been transfected, infected or transformed by the polynucleotide according to claim 10 and / or the vector according to claim 11.

13. The host cell according to claim 12 that is a CAR-T cell.

14. A method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of:- the IL- 15 mutein according to any one of claims 1 to 6, or- the fusion protein according to claim 7, or the immunocytokine according to claim 8 or 9, or- the polynucleotide according to claim 10 or - the vector according to claim 11, or- the host cell according to claim 12 or 13.

15. The method according to claim 14 for treating cancer.

16. A pharmaceutical composition comprising:- the IL- 15 mutein according to any one of claims 1 to 6, or - the fusion protein according to claim 7, or the immunocytokine according to claim 8 or 9, or- the polynucleotide according to claim 10 or- the vector according to claim 11, or- the host cell according to claim 12 or 13.

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