WO2023222886A1

WO2023222886A1 - Antibody-cytokine fusion proteins

Info

Publication number: WO2023222886A1
Application number: PCT/EP2023/063493
Authority: WO
Inventors: Arnaud Foussat; Jemila HOUACINE; Riad ABÈS; William JJ FINLAY; Thibaut De Smedt; Yann Dean
Original assignee: Depth Charge Ltd
Current assignee: Depth Charge Ltd
Priority date: 2022-05-20
Filing date: 2023-05-19
Publication date: 2023-11-23
Anticipated expiration: 2024-11-20

Abstract

The present invention relates to new fusion proteins comprising antibodies or antigen-binding fragments thereof and cytokines, and their use for treating cancer and other diseases.

Description

ANTIBODY-CYTOKINE FUSION PROTEINS

FIELD OF INVENTION

[0001] The present invention relates to fusion proteins comprising antibodies or antigenbinding fragments thereof and cytokines, and to their use for treating cancer and other diseases.

BACKGROUND OF INVENTION

[0002] Cancer is one of the major health issues and is an economic burden for countries. Worldwide, there was an estimated 18.1 million cases and 9.6 million cancer deaths in 2018. Thus, the development of novel and improved cancer therapies is a crucial challenge.

[0003] Cytokines are potent immune-modulating protein molecules that may be used for treating cancer. Indeed, cytokines, such as, for example, IL-2, IL- 15, IL-7 and IL-21, may stimulate the function, survival, and proliferation of natural killer (NK) and T cells that mediate immune responses against tumors. As an example, aldesleukin is a recombinant form of interleukin-2 (IL-2) that was approved for use in the United States for malignant melanoma in 1992, and which is now approved for metastatic renal cell carcinoma and metastatic malignant melanoma.

[0004] However, although cytokine therapy seems promising for treating cancer, it shows several limitations that limit its clinical utility. For example, cytokines have a short half-life, limiting their efficacy in clinical trials. In addition, administration of high doses of cytokines may cause severe side effects, such as, for example, heart problems or neurological disorders. Thus, the development of new safe and efficient cytokines for treating cancer is urgently needed. [0005] The present invention relates to new fusion proteins comprising cytokines, antibodies or antigen-binding fragments thereof and cytokine binding moieties, and their use for treating cancer or other diseases.

SUMMARY

[0006] The present invention relates to a fusion protein comprising: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, and iv. at least one cleavable linker, wherein the at least one cleavable linker links the at least one cytokine binding moiety to any one of the elements of the fusion protein, wherein the cleavage of the at least one cleavable linker releases the at least one cytokine binding moiety, thereby leading to the unmasking of the at least one cytokine.

[0007] In one embodiment, the at least one cleavable linker is cleavable by at least one tumor-associated protease, preferably wherein the at least one tumor-associated protease is selected from the group comprising or consisting of matrix metallopeptidases (MMPs), cathepsins and urokinase-type Plasminogen Activator (uPA), more preferably the at least one cleavable linker comprises the sequence GGGGSPLGLAGFLGS (SEQ ID NO: 30) or the sequence GGGGSPLGLAGGGGS(SEQ ID NO: 129).

[0008] In one embodiment, the at least one cytokine is an interleukin, preferably an interleukin (IL) selected from the group comprising or consisting of IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 12, IL-21, and variants or fragments thereof.

[0009] In one embodiment, the at least one cytokine binding moiety comprises or consists of at least one chain of a native receptor of the at least one cytokine, or a fragment or variant thereof. [0010] In one embodiment, the at least one cytokine is IL- 15 or a fragment or variant thereof, and the at least one cytokine binding moiety comprises or consists of at least one chain of the IL- 15 receptor, or a fragment or variant thereof.

[0011] In one embodiment, the at least one cytokine moiety comprises or consists of the P chain of the IL- 15 receptor, or the domain DI of the P chain of the IL- 15 receptor, or fragments or variants thereof.

[0012] In one embodiment, said fusion protein further comprises an IL-15 receptor a sushi domain or a fragment or variant thereof.

[0013] In one embodiment, said fusion protein further comprises at least one uncleavable linker, preferably wherein the at least one uncleavable linker links the at least one cytokine and/or the IL- 15 receptor a sushi domain to any one of the elements of the fusion protein, more preferably the at least one uncleavable linker is a glycine-serine polymer.

[0014] In one embodiment, the at least one antibody or antigen-binding fragment thereof specifically binds a marker of immune cells, preferably a marker of immune cells selected from the group comprising or consisting of CD25, CD160-TM, PD-1, PDL1, CCR8 and CTLA4, more preferably said at least one antibody or antigen-binding fragment thereof specifically binds CD25.

[0015] In one embodiment, the at least one antibody or antigen-binding fragment thereof specifically binds CD25, and said at least one antibody or antigen-binding fragment thereof comprises one of the following combinations of complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDRs 1-3: SEQ ID NO: 1, 11, 12 and VL-CDRs 1-3: SEQ ID NO: 13, 14, 6, respectively

- VH-CDRs 1-3: SEQ ID NO: 1, 11, 12 and VL-CDRs 1-3: SEQ ID NO: 15, 5, 16, respectively

- VH-CDRs 1-3: SEQ ID NO: 1, 11, 12 and VL-CDRs 1-3: SEQ ID NO: 17, 5, 16, respectively

- VH-CDRs 1-3: SEQ ID NO: 1, 11, 12 and VL-CDRs 1-3: SEQ ID NO: 18, 5, 6, respectively - VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 20, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 21, 14, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 22, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 23, 5, 6, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 18, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 24, 14, 6, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 25, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 21, 5, 6, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 26, 5, 6, respectively

- VH-CDRs 1 -3 : SEQ ID NO : 1 , 27, 19, and VL-CDRs 1 -3 : SEQ ID NO : 23 , 5, 16, respectively

- VH-CDRs 1 -3 : SEQ ID NO : 1 , 27, 19, and VL-CDRs 1 -3 : SEQ ID NO : 21 , 14, 16, respectively

- VH-CDRs 1 -3 : SEQ ID NO : 1 , 27, 19, and VL-CDRs 1 -3 : SEQ ID NO : 24, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1,28, 19, and VL-CDRs 1-3: SEQIDNO: 24, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1,29, 19, and VL-CDRs 1-3: SEQIDNO: 25, 5, 6, respectively.

[0016] The present invention further relates to a nucleic acid encoding a fusion protein as defined hereinabove. [0017] The present invention further relates to an expression vector comprising a nucleic acid as defined hereinabove.

[0018] The present invention further relates to a pharmaceutical composition comprising a fusion protein as defined hereinabove, a nucleic acid as defined hereinabove, or an expression vector as defined hereinabove, and a pharmaceutically acceptable excipient.

[0019] The present invention further relates to the fusion protein as defined hereinabove, the nucleic acid as defined hereinabove, the expression vector as defined hereinabove, or the pharmaceutical composition as defined hereinabove, for use as a medicament.

[0020] The present invention further relates to the fusion protein as defined hereinabove, the nucleic acid as defined hereinabove, the expression vector as defined hereinabove, or the pharmaceutical composition as defined hereinabove, for use in treating a cancer or an infectious disease in a subject in need thereof.

DEFINITIONS

[0021] In the present invention, the following terms have the following meanings:

[0022] “About”, preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0023] “Affinity” and “avidity” are used to defined the strength of an antibody-antigen complex. Affinity measures the strength of interaction between an epitope and an antigen binding site on an antibody (or fragment thereof). It may be expressed by an affinity constant KA or by a dissociation constant KD. Avidity (or functional affinity) gives a measure of the overall strength of an antibody-antigen complex. It may depend on different parameters, including in particular, the affinity of the antibody or antigenbinding fragment thereof for an epitope, (ii) the valency of both the antibody and the antigen and (iii) structural arrangement of the parts that interact. [0024] “Antibody” and “immunoglobulin”, as used herein, may be used interchangeably and refer to a protein having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. “Antibodies” refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g., human CD25). The term “anti-hCD25 antibodies” is used herein to refer to antibodies which exhibit immunological specificity for human CD25 protein. As explained elsewhere herein, “specificity” for human CD25 (hCD25) does not exclude cross-reaction with species homologues of hCD25, such as, for example, with simian CD25. Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood. The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. Although the following discussion will generally be directed to the IgG class of immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight of about 23 kDa, and two identical heavy chains of molecular weight of about 53-70 kDa. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. The light chains of an antibody are classified as either kappa (K) or lambda (X). Each heavy chain class may be bonded with either a K or light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” regions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma (y), mu (p), alpha (a), delta (8) or epsilon (a) with some subclasses among them e.g., yl -y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgD or IgE, respectively. The immunoglobulin subclasses or “isotypes” (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc.) are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the present invention. As indicated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the light chain variable domain (VL domain) and heavy chain variable domain (VH domain) of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site presents at the end of each arm of the “Y”. More specifically, the antigen binding site is defined by three complementarity determining regions (CDRs) on each of the VH and VL chains.

[0025] “Antigen-binding fragment”, as used herein, refers to a part or region of an antibody which comprises fewer amino acid residues than the whole antibody. An “antigen-binding fragment” binds antigen and/or competes with the whole antibody from which it derives for antigen binding (e.g., specific binding to human CD25). Antigenbinding fragments encompasses, without any limitation, single chain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab)’2, Fd, defucosylated antibodies, diabodies, triabodies and tetrabodies. This term may also include unibodies, nanobodies, and domain antibodies.

[0026] “CD160” has its general meaning in the art and refers to CD 160 molecule. Three CD 160 isoforms exist: the CD160-TM isoform, the CD 160 GPI-anchored isoform and the soluble CD 160 isoform. CD160-GPI is expressed by intestinal intraepithelial T lymphocytes and by a minor subset of circulating lymphocytes including NK cells, TCRyb and cytotoxic effector CD8^bnghtCD28- T lymphocytes (ANUMANTHAN et al., 1998, J Immunol; 161 :2780-2790; MAIZA et al., J. Exp. Med., vol. 178, p: 1121-1126, 1993). The CD 160 transmembrane isoform (“CD160-TM”) is described in Giustiniani J et al. (J Immunol. 2009 Jan 1 ; 182(1):63-71.) as well as in the international patent application W02008155363 and is characterized by the amino acid sequence as set forth in SEQ ID NO: 123. The extracellular domain of the CD160-TM isoform may be defined by the amino acid sequence ranging from the amino acid residue at position 26 to the amino acid residue at position 189 in SEQ ID NO: 123. The CD 160 GPI-anchored isoform (“CD160-GPI”) is described in Nikolova M. et al. (Int Immunol. 2002 May;14(5):445-51.) as well as in the international patent application W02006015886 and is characterized by the amino acid sequence as set forth in SEQ ID NO: 124 fused to a GPI anchor at the C terminus end. The CD 160 soluble isoform is described in Giustiniani J. et al. (J Immunol. 2007 Feb l;178(3): 1293-300) and is characterized by the amino acid sequence as set forth in SEQ ID NO: 124. In SEQ ID NO: 123-124, amino acids 1-25 correspond to a signal peptide, and may consequently be absent from the expressed protein.

SEQ ID NO: 123: CD160-TM isoform

MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEEAEG FVVFLCKDRSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTPLHSGTY QCCARSQKSGIRLQGHFFSILFTETGNYTVTGLKQRQHLEFSHNEGTLSSGFLQE KVWVMLVTSLVALQGMSKRAVSTPSNEGAIIFLPPWLFSRRRRLERMSRGREK C YS SPGYPQES SNQFH

SEQ ID NO: 124: CD160 soluble isoform MLLEPGRGCCALAILLAIVDIQSGGCINITSSASQEGTRLNLICTVWHKKEEAEG FVVFLCKDRSGDCSPETSLKQLRLKRDPGIDGVGEISSQLMFTISQVTPLHSGTY QCCARSQKSGIRLQGHFFSILFTETGNYTVTGLKQRQHLEFSHNEGTLSS

[0027] “CD25” refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The Interleukin-2 receptor alpha chain (also called CD25) protein is encoded by the IL2RA gene. Two forms of the IL-2 receptor were described: the first one comprising the alpha subunit (CD25), the beta subunit (CD122) and the gamma subunit (CD132), and the second one comprising only the beta and gamma subunits (i.e., CD122 and CD 132). The term encompasses "full-length" or unprocessed CD25 as well as any form of CD25 that results from processing in the cell. The term also encompasses naturally occurring variants of CD25 (e.g., splice variants or allelic variants). In certain embodiments CD25 is human CD25. For example, CD25 is expressed by activated T lymphocytes and activated B lymphocytes responding to antigen or mitogen stimulation. CD25 is also expressed by regulatory T cells (CD25^highFoxP3⁺ regulatory T cells). In one embodiment, CD25 refers to human CD25 (Uniprot accession number P01589, SEQ ID NO: 122). SEQ ID NO: 122

MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECK RGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKT TEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRA LHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETS CLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQR KSRRTI

[0028] “CDR” or “complementarity determining region” means the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), or a combination thereof. More recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. 27: 209-212 1999). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the "location" of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues may be readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. Correspondence between the Kabat numbering and the IMGT unique numbering system is also well known to one skilled in the art (e.g., Lefranc et al., supra). Thus, in one embodiment, by CDR regions or CDR, it is intended to indicate the hypervariable regions of the heavy and light chains of the immunoglobulins as defined by IMGT® numbering system (e.g. Lefranc et al., supra). [0029] Cleavable linker” refers to a linker that responds to physiological stimuli such as high glutathione concentrations, or enzymatic including proteolytic cleavage. In one embodiment, the cleavable linker responds to proteolytic cleavage.

[0030] “Cytokine” refers to a secreted polypeptide, or fragment or variant thereof, that modulates the activity of cells, particularly cells of the immune system. Examples of cytokines include, for instance, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. The term encompasses any cytokine protein, or a functional fragment or variant thereof. The term encompasses any native cytokine from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice), unless otherwise indicated. The term encompasses an unprocessed form of the cytokine as well as any form of the cytokine that results from processing in a cell. The term also encompasses naturally occurring variants of a cytokine. The term also encompasses non-naturally occurring variants of a cytokine, such as variants caused by substitution, addition, and/or deletion of at least one amino acid.

[0031] “Cytokine receptor”: refers to any receptor within the art that binds to one or more cytokine(s) including, but not limited to, receptors of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-P), IFN-y (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, AP03L, CD256, CD257, CD258, TL1, AITRL, EDAI, interferon (IFN)-a (alpha), IFN-p (beta), IFN-y (gamma), growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), and thrombopoietin (TPO). Some cytokine receptors function, in whole or in part, as heteromeric complexes of more than one subunit, or as homomeric complexes. In particular, the term “native cytokine receptor” encompasses receptors that bind to one or more cytokine(s), and that are from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice), unless otherwise indicated. [0032] “Diabodies”, as used herein, refer to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL such that inter-chain but not intra-chain pairing of the variable domains is achieved, resulting in a bivalent fragment, /.<?., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL of the two antibodies are present on different polypeptide chains. Diabodies are described, for example, in patent EP0404097 or patent application WO1993011161.

[0033] “Domain antibodies” refer to the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies.

[0034] “Effector T cells” refer to a group of cells that includes several T cell types (e.g., CD4⁺ and CD8⁺ T cells). It includes helpers T cells (Th cells) that help other leukocytes in immunologic processes, including maturation of B cells into plasma cells and memory B cells and cytotoxic T cells (Tc cells, CTLs, T-killer cells, killer T cells) that destroy virus-infected cells and tumor cells, and are also implicated in transplant rejection.

[0035] “Epitope” refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody or antigen-binding fragment thereof binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear (or sequential) or conformational, /.<?., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.

[0036] “Fab” refers to a monovalent fragment containing the following regions: VH, VL, CHI and CL, linked by an intramolecular disulfide bond. As used herein, F(ab')2 refers to a fragment containing two antigen-binding regions joined by disulfides bonds. As used herein, Fab’ refers to a fragment obtained by the reduction of F(ab')2 fragments.

[0037] “Fd fragment” refers to the heavy chain of the Fab fragment, comprising the VH and CHI regions. [0038] “Fv”, as used herein, refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one VH and one VL in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the heavy and light chain) that contribute to antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0039] “Heavy chain region” includes amino acid sequences derived from the constant domains of an immunoglobulin heavy chain. A protein comprising a heavy chain region comprises at least one of a CHI domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In an embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention may comprise the Fc region of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention lacks at least a region of a constant domain (e.g., all or part of a CH2 domain). In certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one embodiment, the heavy chain region comprises a fully human hinge domain. In other embodiments, the heavy chain region comprises a fully human Fc region (e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin). In certain embodiments, the constituent constant domains of the heavy chain region are from different immunoglobulin molecules. For example, a heavy chain region of a protein may comprise a CH2 domain derived from an IgGl molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are chimeric domains comprising regions of different immunoglobulin molecules. For example, a hinge may comprise a first region from an IgGl molecule and a second region from an IgG3 or IgG4 molecule. In some embodiments, the constant domains of the heavy chain region may be modified such that they vary in amino acid sequence from the naturally occurring (wild-type) immunoglobulin molecule. That is, the antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention may comprise alterations or modifications to one or more of the heavy chain constant domains (CHI, hinge, CH2 or CH3) and/or to the light chain constant domain (CL). Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.

[0040] “Identity” or “identical”, when used herein in a relationship between the sequences of two or more amino acid sequences, or of two or more nucleic acid sequences, refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (/.< ., “algorithms”). Identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carillo etal., 1988. SIAMJApplMath. 48(5): 1073-82. Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, University of Wisconsin, Madison, WI; Devereux etal., 1984. Nucleic Acids Res. 12(1 Pt l):387-95), BLASTP, BLASTN, and FASTA (Altschul etal., 1990. J Mol Biol. 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may also be used to determine identity.

[0041] “Immune cells” refer to any various cells that function in the immune system, to protect against agents of infection. In one embodiment, this term includes immune regulatory cells such as Regulatory T cells (Treg), B regulatory cells (Breg) and Myeloid derived suppressor cells (MDSC), M2 -type macrophages and immune effector cells such as T cells, including CD4+ T cells and CD8+ T cells; B cells; natural killers (NK) cells; natural killer T (NKT) cells; dendritic cells, mast cells and Ml macrophages.

[0042] “Interleukin-2” or “IL-2” refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2 (e.g., splice variants or allelic variants). In one embodiment, IL-2 is human IL-2, having the sequence SEQ ID NO: 32.

SEQ ID NO: 32

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0043] “Interleukin-4” or “IL-4” refers to any native IL-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-4 as well as any form of IL-4 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-4 (e.g., splice variants or allelic variants). In one embodiment, IL-4 is human IL-4, having the sequence SEQ ID NO: 33 (UniProtKB - Q5FC01).

SEQ ID NO: 33

MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKNTTEKETFCR AATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANQ STLENF LERLKT IM REK YSKC S S [0044] “Interleukin-7” or “IL-7” refers to any native IL-7 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-7 as well as any form of IL-7 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-7 (e.g., splice variants or allelic variants). In one embodiment, IL-7 is human IL-7, having the sequence SEQ ID NO: 34 (UniProtKB - A8K673).

SEQ ID NO: 34

MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDSKQYESVLMVSIDQLLDSMKEI GSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVS EGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQE IKTCWNKILMGTKEH

[0045] “Interleukin-9” or “IL-9” refers to any native IL-7 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-9 as well as any form of IL-9 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-9 (e.g., splice variants or allelic variants). In one embodiment, IL-9 is human IL-9, having the sequence SEQ ID NO: 35 (UniProtKB - Q6NZ74).

SEQ ID NO: 35

MLLAMVLTSALLLCSVAGQGCPTLAGILDINFLINKMQEDPASKCHCSANVTSC PCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSVEVLKNNKCPYFSC EQPCNQTTAGNALTFLKSLLEIFQKEKMRGMRGKI

[0046] “Interleukin- 15” or “IL-15” refers to any native IL-15 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL- 15 as well as any form of IL-15 that results from processing in the cell. The term also encompasses naturally occurring variants of IL- 15 (e.g., splice variants or allelic variants). In one embodiment, IL-15 is human IL-15, having the sequence SEQ ID NO: 31.

SEQ ID NO: 31 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG

DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI

NTS

[0047] “Interleukin- 15 receptor” or “IL- 15 receptor” refers to any native IL- 15 receptor from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The IL-15 receptor is a heteromultimeric complex formed by the combination of the two subunits IL-15RP chain (also known as IL-2RP chain or CD 122) and IL-15Ry chain (also known as IL-2RY chain or CD132) or the combination of the three subunits IL-15Ra chain, IL-15RP chain and fL-15Ry chain. In one embodiment, the IL-15 receptor is a human IL-15 receptor.

[0048] “Interleukin-12” or “IL-12” refers to any native IL-12 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-12 as well as any form of IL-12 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-12 (e.g., splice variants or allelic variants). IL-12 is a heterodimeric cytokine encoded by two separate genes, IL-12 A (p35) and IL-12B (p40) in humans. In one embodiment, IL-12 is human IL-12.

[0049] “Interleukin-21” or “IL-21” refers to any native IL-21 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-21 as well as any form of IL-21 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-21 (e.g., splice variants or allelic variants). In one embodiment, IL-21 is human IL-21, having the sequence SEQ ID NO: 36 (UniProtKB - A0A224B028).

SEQ ID NO: 36

MRS SPGNMERIVICLMVIFLGTLVHKS S SQGQDRHMIRMRQLIDIVDQLKNYVN DLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPS TNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS [0050] “Mammal” refers to any mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0051] “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, /.< ., the individual antibodies comprised in the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies or antigen-binding fragment thereof comprised in a fusion protein according to the present invention may be prepared by the hybridoma methodology first described by Kohler et al., 1975. Nature. 256(5517):495-7, or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (Patent US4, 816, 567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et a!., 1991. Nature. 352(6336): 624-8 and Marks etal., 1991. J Mol Biol. 222(3): 581-97, for example.

[0052] “Nanobodies” refer to antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy chain antibodies (Muyldermans, 2013. Annu Rev Biochem. 82:775-97). These heavy chain antibodies may contain a single variable domain (VHH) and two constant domains (CH2 and CH3).

[0053] “Regulatory T cell" or "Treg cell” refers to a specialized type of T cells, in particular of CD4⁺ T cell, that can suppress the responses of other T cells. Treg cells are generally characterized by expression of CD4, the a-subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (Foxp3) and play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. More recently, CD8 Tregs have also been described.

[0054] “Single chain antibody”, as used herein, refers to any antibody or fragment thereof that is a protein having a primary structure comprising or consisting of one uninterrupted sequence of contiguous amino acid residues, including without limitation (1) single-chain Fv molecules (scFv); (2) single chain proteins containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety; and (3) single chain proteins containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety.

[0055] “Single-chain Fv”, also abbreviated as “sFv” or “scFv”, refers to antibody fragments that comprise the VH and VL antibody domains connected into a single amino acid chain. Preferably, the scFv amino acid sequence further comprises a peptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0056] “Specifically bind” or “specific to” refers to an antibody or antigen-binding fragment thereof that reacts at a detectable level with an antigen (e.g., CD25), preferably with an affinity constant (KA) of greater than or equal to about 10⁶ M'¹, preferably greater than or equal to about 10⁷ M^-1, 10⁸ M'¹, 5xl0⁸M^-1, 10⁹ M'¹ , 5xl0⁹ M'¹ or more. Affinity of an antibody or antigen-binding fragment thereof for its cognate antigen is also commonly expressed as an equilibrium dissociation constant (KD). An antibody or antigen-binding fragment thereof is said to be “immunospecific”, “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with said antigen (e.g., CD25), preferably with a KD of less than or equal to 10'⁶ M, preferably less than or equal to 10'⁷ M, 5.1O'⁸ M, 10'⁸ M, 5.1O'⁹ M, IO'⁹ M or less.

[0057] “Subject”, as used herein, refers to a mammal, preferably a human. In one embodiment, a subject may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease.

[0058] “Therapeutically effective amount” refers to the level or amount of an antibody as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.

[0059] “Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In one embodiment, a subject is successfully "treated" for a cancer or an infectious disease if, after receiving a therapeutic amount of an antibody according to the present invention, the subject shows at least one of the following: reduction in the number of cancer cells (or tumor size) or pathogenic cells; reduction in the percent of total cells that are cancerous or pathogenic; relief to some extent of one or more of the symptoms associated with the cancer or the infectious disease to be treated; reduced morbidity and mortality; and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0060] “Unibodies” refer to an antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies. [0061] “Variable” refers to the fact that certain regions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called “hypervariable loops” in each of the VL domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the VX light chain domain are referred to herein as LI (X), L2 (X) and L3 (X) and may be defined as comprising residues 24-33 (L1(X), consisting of 9, 10 or 11 amino acid residues), 49-53 L2 (X), consisting of 3 residues) and 90-96 (L3(X), consisting of 6 residues) in the VL domain (Morea et al., 2000. Methods. 20(3):267-79). The first, second and third hypervariable loops of the VK light chain domain are referred to herein as 1(K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1(K), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et al., supra). The first, second and third hypervariable loops of the VH domain are referred to herein as Hl, H2 and H3 and may be defined as comprising residues 25-33 (Hl, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea etal., supra). Unless otherwise indicated, the terms LI, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both VK and VX isotypes. The terms Hl, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including gamma (y), mu (p), alpha (a), delta (8) or epsilon (a). The hypervariable loops LI, L2, L3, Hl, H2 and H3 may each comprise part of a “complementarity determining region” or “CDR”, as defined hereinabove.

DETAILED DESCRIPTION

[0062] The present invention relates to a fusion protein comprising: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, and iv. at least one cleavable linker.

[0063] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is selected from the group comprising or consisting of interferons (IFN), interleukins (IL), tumor necrosis factors (TNF), chemokines, colonystimulating factors (CSF), and variants or fragments thereof.

[0064] Examples of cytokines include, without limitation, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL- 30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-P), IFN-y (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, AP03L, CD256, CD257, CD258, TL1, AITRL, EDAI, interferon (IFN)-a (alpha), IFN-p (beta), IFN-y (gamma), growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), and thrombopoietin (TPO).

[0065] In one embodiment, the at least one cytokine comprised in a fusion protein according to the present invention is an IL, or a variant or fragment thereof. Examples of IL include, without limitation, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL- 23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and IL-36.

[0066] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is an IL selected from the group comprising or consisting of IL-15, IL-2, IL-4, IL-7, IL-9, IL-12 and IL-21, and variants or fragments thereof.

[0067] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL- 15, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL- 15, or a variant or fragment thereof.

[0068] In one embodiment, the at least one cytokine comprises or consists of the amino acid sequence SEQ ID NO: 31, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 31.

[0069] Examples of IL- 15 variants are provided in the patent application W02018/071918 and include, without limitation, IL-15 comprising one or more of the following amino acid substitutions: N1D, N4D, D30N, D61N, E64Q, Q108E, N1D/D61N, N1D/E64Q, N1D/N65D, N1D/Q108E, N4D/D61N, N4D/E64Q, N4D/N65D, D8N/D61N, D8N/E64Q, D30N/N65D, D30N/Q108E, D61N/E64Q, E64Q/N65D, E64Q/Q108E, N65D/Q108E, N1D/N4D/D8N, N1D/N4D/N65D, N4D/D61N/N65D, N4D/D61N/E64Q/Q108E, and D30N/E64Q/N65D.

[0070] Examples of IL-15 variants are provided in the patent application WO2021/119516 and include, without limitation, IL-15 comprising one or more of the following amino acid substitutions: D30T, V31Y, H32E, S58D, S58G, S58H, S58R, S58Q, S58I, S58P, T61D, V63F, V63A, V63K, V63R, I67V, I68F, I68H, I68D, I68K, I68Q, I68G, Q108A, Q108M, Q108S, Q108E and Q108K.

[0071] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL-2, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL-2, or a variant or fragment thereof.

[0072] In one embodiment, the at least one cytokine comprises or consists of the amino acid sequence SEQ ID NO: 32, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 32.

[0073] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL-4, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL-4, or a variant or fragment thereof. [0074] In one embodiment, the at least one cytokine comprises or consists of the amino acid sequence SEQ ID NO: 33, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 33.

[0075] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL-7, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL-7, or a variant or fragment thereof.

[0076] In one embodiment, the at least one cytokine comprises or consists of the amino acid sequence SEQ ID NO: 34, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 34.

[0077] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL-9, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL-9, or a variant or fragment thereof.

[0078] In one embodiment, the at least one cytokine comprises or consists of the amino acid sequence SEQ ID NO: 35, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 35.

[0079] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL-12, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL-12, or a variant or fragment thereof.

[0080] In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is IL-21, or a variant or fragment thereof. In one embodiment, the at least one cytokine comprised in the fusion protein according to the present invention is a human IL-21, or a variant or fragment thereof.

[0081] In one embodiment, the at least one cytokine comprises or consists of the amino acid sequence SEQ ID NO: 36, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 36. [0082] In one embodiment, the fusion protein according to the present invention comprises one, two, three or more cytokine(s) as defined hereinabove. In one embodiment wherein the fusion protein according to the present invention comprises two or more cytokines, said cytokines are identical. In one embodiment wherein the fusion protein according to the present invention comprises two or more cytokines, said cytokines are different.

[0083] In one embodiment, the at least one cytokine binding moiety of the fusion protein according to the present invention has a weaker affinity for the at least one cytokine as compared to the native receptor of the at least one cytokine.

[0084] In one embodiment, the at least one cytokine binding moiety of the fusion protein according to the present invention has a stronger affinity for the at least one cytokine as compared to the native receptor of the at least one cytokine.

[0085] Methods for comparing the binding affinity of the at least one cytokine binding moiety and of the native cytokine receptor to a cytokine are well known in the art, and include, without limitation, measuring binding kinetics of the interactions or performing competition experiments.

[0086] Affinity of a protein is commonly expressed as an equilibrium dissociation constant (KD). Thus, in one embodiment, the binding kinetics, such as the KD, of the at least one cytokine binding moiety and of the native cytokine receptor to the cytokine are determined by surface plasmon resonance (SPR, e.g., using BIAcore®).

[0087] In one embodiment, the at least one cytokine binding moiety has a weaker affinity to the at least one cytokine as compared to the native cytokine receptor when the KD of the interaction between the at least one cytokine binding moiety and the at least one cytokine is higher than the KD of the interaction between the native cytokine receptor and the at least one cytokine.

[0088] In one embodiment, the at least one cytokine binding moiety has a stronger affinity to the at least one cytokine as compared to the native cytokine receptor when the KD of the interaction between the at least one cytokine binding moiety and the at least one cytokine is lower than the KD of the interaction between the native cytokine receptor and the at least one cytokine.

[0089] In one embodiment, the native cytokine receptor is a native full-length receptor of a cytokine. Thus, in one embodiment, the native cytokine receptor includes any native receptor of a cytokine as defined hereinabove.

[0090] Examples of cytokine receptors are well-known by the skilled artisan in the art, and include, without limitation, type I cytokine receptors, type II cytokine receptors, immunoglobulin (Ig) superfamily, tumor necrosis factor receptor family, chemokine receptors and TGF-beta receptor family.

[0091] In one embodiment, when the at least one cytokine is IL- 15, IL-2, IL-4, IL-7, IL- 9 IL- 12 or IL-21, the native cytokine receptor is IL- 15 receptor, IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-12 receptor or IL-21 receptor, respectively. In one embodiment, when the at least one cytokine is human IL- 15, IL-2, IL-4, IL-7, IL-9, IL- 12 or IL-21, the native cytokine receptor is human IL- 15 receptor, IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL- 12 receptor or IL-21 receptor, respectively.

[0092] In one embodiment, when the at least one cytokine is IL-2, the native cytokine receptor is the heterotrimeric IL-2 receptor composed of the three subunits: IL-2Ra chain (also known as CD25, Tac antigen), IL-2RP chain (also known as CD 122) and IL-2Ry chain (also known as CD 132).

[0093] In one embodiment, when the at least one cytokine is IL-15, the native cytokine receptor is the heterotrimeric IL-15 receptor composed of the three subunits: IL-15Ra chain, IL-15RP chain (also known as CD 122) and IL-lSRy chain (also known as CD 132).

[0094] In one embodiment, the at least one cytokine binding moiety comprises or consists of at least one chain of a native receptor of the at least one cytokine, or a fragment or variant thereof.

[0095] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of at least one chain of the IL- 15 receptor, or a fragment or variant thereof. [0096] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of the P chain of the IL-15 receptor, or a fragment or variant thereof.

[0097] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 37, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 37.

SEQ ID NO: 37

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLP VSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLR LMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTL KQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD T

[0098] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of the domain DI of the P chain of the IL- 15 receptor, or a fragment or variant thereof.

[0099] In one embodiment, the at least one cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 38 or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 38.

SEQ ID NO: 38

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLP VSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFEN

[0100] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of the y chain of the IL- 15 receptor, or a fragment or variant thereof. [0101] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of a fragment of the y chain of the IL- 15 receptor, or a variant thereof.

[0102] In one embodiment, when the at least one cytokine is IL-15, the at least one cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 39, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 39.

SEQ ID NO: 39

MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLP LPEVQCF VFNVEYMNCTWNS S SEPQPTNLTLHYW YKNSDNDK VQKC SHYLF S EEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTL HKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVD GQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVG SMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQ PDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0103] In one embodiment, the at least one cytokine binding moiety comprises or consists of an antibody or an antigen-binding fragment thereof that specifically binds the at least one cytokine.

[0104] In one embodiment, the fusion protein according to the present invention comprises one, two, three or more cytokine binding moiety(ies) as defined hereinabove. In one embodiment wherein the fusion protein according to the present invention comprises two or more cytokine binding moieties, said cytokine binding moieties are identical. In one embodiment wherein the fusion protein according to the present invention comprises two or more cytokine binding moieties, said cytokine binding moieties are different.

[0105] In one embodiment, the at least one cytokine binding moiety reduces the activity of the at least one cytokine or the binding or the affinity of the at least one cytokine to its native receptor. As an example, when bound to the at least one cytokine, the at least one cytokine binding moiety blocks, occludes, decreases or prevents the activity of the at least one cytokine or the binding or the affinity of the at least one cytokine to its native receptor.

[0106] In one embodiment, the binding of the at least one cytokine binding moiety to the at least one cytokine reduces the binding or the affinity of the at least one cytokine to its native receptor by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more.

[0107] Methods for assessing the binding or affinity of proteins are defined hereinabove.

[0108] In one embodiment, the binding of the at least one cytokine binding moiety to the at least one cytokine reduces the activity of the at least one cytokine by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more.

[0109] Methods for assessing the activity of cytokines are well-known by the skilled artisan and will depend on the cytokine comprised in the fusion protein according to the present invention.

[0110] Methods for measuring the IL-2 signaling are well known in the art and comprise, for example, the measurement of the induction of IL-2 receptor signaling (e.g., by detection of phosphorylated STAT5a), the measurement of the induction of T cell proliferation (e.g., by detection of Ki-67 using in particular CellTrace™ Cell Proliferation Kits, by direct assessment of T cell proliferation in the presence of IL-2, in MLR experiments (comprising, for example, the activation of cells with CD3 and CD28 in the presence of IL-2), or using cell lines that depend on IL-2 to proliferate, such as, for example CTLL2 cell line) and/or the measurement of an up-regulation of expression of activation markers (such as e.g., CD25, CD69, cytotoxic molecules, such as, for example, granzyme B, and the like).

[0111] Methods for measuring the IL- 15 signaling are well known in the art and comprise, for example, the measurement of the induction of IL- 15 receptor signaling by measuring phosphorylated STAT5 expression in cells, such as, for example, CD4+ T cell, CD8+ T cells or NK cells, or by measuring the induction of IL- 15 -induced proliferation of cells, such as, for example, CD8+ T cells or NK cells (e.g. by CellTrace Far Red Cell Proliferation Kit). Examples of such methods are provided in the Example part.

[0112] In one embodiment, the at least one cleavable linker links the at least one cytokine binding moiety to any one of the elements of the fusion protein.

[0113] Thus, in one embodiment, the fusion protein of the present invention comprises: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, and iv. at least one cleavable linker, wherein the at least one cleavable linker links the at least one cytokine binding moiety to any one of the elements of the fusion protein.

[0114] In one embodiment, the cleavage of the at least one cleavable linker releases the at least one cytokine binding moiety, thereby leading to the unmasking of the at least one cytokine.

[0115] In one embodiment, the at least one cleavable linker comprised in the fusion protein according to the present invention is a protease-cleavable linker. Thus, in one embodiment, the at least one cleavable linker comprised in the fusion protein according to the present invention responds to proteolytic cleavage.

[0116] In one embodiment, the at least one cleavable linker is cleavable by tumor- associated proteases. Examples of tumor-associated proteases include, without limitation, matrix metallopeptidases (MMPs), cathepsins (including cathepsin B) and urokinase-type Plasminogen Activator (uPA).

[0117] Thus, in one embodiment, the at least one cleavable linker is cleavable by tumor- associated proteases selected from the group comprising matrix metallopeptidases (MMPs), cathepsins and urokinase-type Plasminogen Activator (uPA). [0118] In one embodiment, the at least one cleavable linker comprises at least one protease recognition site. In one embodiment, the at least one cleavable linker comprises two, three or more protease recognition sites.

[0119] In one embodiment, the at least one cleavable linker is a glycine-serine polymer, comprising at least one recognition site.

[0120] Example of glycine-serine polymers include, without limitation, (GS)n, (GSGGS)n(SEQ ID NO: 97), (GGGGS)n(SEQ ID NO: 98), and (GGGS)n(SEQ ID NO: 99), where n is an integer of at least one (and generally from 1 to 5), as well as any peptide sequence that allows for recombinant attachment of the at least one cytokine binding moiety to any one of the elements of the fusion protein with sufficient length and flexibility to allow each element to retain its biological function.

[0121] In one embodiment, the at least one cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PX(GGGGS)_q]t (SEQ ID NO: 100), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3, and wherein X is at least one protease recognition site.

[0122] In one embodiment, said cleavable linker may further comprise one or several amino acid residues, such as a serine residue, after the at least one protease recognition site. Thus, in one embodiment, the at least one cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PXS(GGGGS)_q]t (SEQ ID NO: 101), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3, and wherein X is at least one protease recognition site.

[0123] In one embodiment, the at least one protease recognition site is a peptide motif cleaved by MMP, cathepsin B or uPA.

[0124] Examples of MMP include, without limitation, MMP2, MM9, MMP8, MMP 12, MMP13, and MMP14. In one embodiment, the at least one protease recognition site is a peptide motif cleaved by a MMP selected from the group comprising or consisting of MMP2, MM9, MMP8, MMP 12, MMP 13, and MMP 14, preferably MMP8. [0125] Examples of peptide motifs cleaved by MMP include, without limitation, PLGLA (SEQ ID NO: 102) (cleaved by MMP-2/8/9); PLGVR (SEQ ID NO: 103) (cleaved by MMP-2/9/13); PLGIAG (SEQ ID NO: 104) (cleaved by MMP-2); GPLGV (SEQ ID NO: 105) (cleaved by MMP-2/9/13), GPLGVRG (SEQ ID NO: 106) (cleaved by MMP-2, -9, -13), SGFIANPVTA (cleaved by MMP14) (SEQ ID NO: 126) and PLGL (cleaved by MMP8) (SEQ ID NO: 127).

[0126] Examples of peptide motifs cleaved by cathepsin B include, without limitation, FKC and GFLG (SEQ ID NO: 108).

[0127] Examples of peptide motifs cleaved by uPA include, without limitation, GGGRR (SEQ ID NO: 109) and SGRSA (SEQ ID NO: 110).

[0128] In one embodiment, the at least one cleavable linker comprised in the fusion protein according to the present invention comprises several identical peptide motifs, as defined hereinabove.

[0129] In one embodiment, the at least one cleavable linker comprised in the fusion protein according to the present invention comprises several different peptide motifs, which are cleaved by the same protease, as defined hereinabove.

[0130] In one embodiment, the at least one cleavable linker comprised in the fusion protein according to the present invention comprises several different peptide motifs, which are cleaved by different proteases, as defined hereinabove. Thus, in one embodiment, the at least one cleavable linker comprised in the fusion protein according to the present invention comprises several different peptide motifs cleaved by MMP, cathepsin B and/or uPA.

[0131] In one embodiment, the at least one cleavable linker is a glycine-serine polymer, comprising PLGLA (SEQ ID NO: 102) and/or PLGL (SEQ ID NO: 127).

[0132] In one embodiment, the at least one cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PPLGLA(GGGGS)_q]t (SEQ ID NO: 125), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3. [0133] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence GGGGSPLGLAGGGGS (SEQ ID NO: 129).

[0134] In one embodiment, the at least one cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PPLGLAGFLGS(GGGGS)_q]t (SEQ ID NO: 111), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3.

[0135] In one embodiment, the at least one cleavable linker comprises the sequence GGGGSPLGLAGFLGS (SEQ ID NO: 30).

[0136] In one embodiment, the at least one cleavable linker is selected from the group comprising or consisting of GGGGSPLGLAGFLGS (SEQ ID NO: 30), GGGGSPLGLAGFLGS(GGGGS)i (SEQ ID NO: 113) and

GGGGSPLGLAGFLGS(GGGGS)₂ (SEQ ID NO: 114), and GGGGSPLGLAGGGGS (SEQ ID NO: 129).

[0137] In one embodiment, the fusion protein according to the present invention further comprises an IL-15 receptor a sushi domain, or a variant or fragment thereof.

[0138] Thus, the present invention further relates to a fusion protein as described hereinabove comprising: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, preferably IL- 15 or a variant or fragment thereof, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, iv. at least one cleavable linker, and v. an IL- 15 receptor a sushi domain, or a variant or fragment thereof.

[0139] In one embodiment, the fusion protein according to the present invention comprises an IL- 15 receptor a sushi domain comprising or consisting of the sequence SEQ ID NO: 107, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 107. SEQ ID NO: 107

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVA

HWTTPSLKCIR

[0140] In one embodiment, the fusion protein according to the present invention further comprises at least one uncleavable linker.

[0141] Thus, the present invention further relates to a fusion protein as described hereinabove comprising: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, iv. at least one cleavable linker, and v. at least one uncleavable linker.

[0142] Thus, the present invention further relates to a fusion protein as described hereinabove comprising: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, preferably IL- 15 or a variant or fragment thereof, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, iv. at least one cleavable linker, v. an IL- 15 receptor a sushi domain, or a variant or fragment thereof, vi. and at least one uncleavable linker.

[0143] In one embodiment, the fusion protein according to the present invention comprises one, two, three or more uncleavable linker(s).

[0144] In one embodiment, the at least one uncleavable linker links the at least one cytokine to any one of the elements of the fusion protein. In one embodiment, the at least one uncleavable linker links the at least one cytokine to the at least one antibody or antigen-binding fragment thereof or to the IL- 15 receptor a sushi domain. [0145] In one embodiment, the at least one uncleavable linker links the IL-15 receptor a sushi domain to any one of the elements of the fusion protein. In one embodiment, the at least one uncleavable linker links the IL-15 receptor a sushi domain to the at least one antibody or antigen-binding fragment thereof or to the at least one cytokine.

[0146] In one embodiment, the at least one uncleavable linker is a glycine-serine polymer. Examples of glycine-serine polymers are provided hereinabove.

[0147] In one embodiment, the at least one uncleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p and q independently range from 0 to 8, wherein t ranges from 1 to 3.

[0148] In one embodiment, the at least one uncleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p ranges from 1 to 6, q ranges from 0 to 4, wherein t ranges from 1 to 3, preferably wherein t equals 1.

[0149] In one embodiment, the at least one uncleavable linker is selected from the group comprising or consisting of (GGGGS)₂GGGG (SEQ ID NO: 116), (GGGGS)₃GGGG (SEQ ID NO: 117), (GGGGS)eGG (SEQ ID NO: 118), (GGGGS)₃ (SEQ ID NO: 119), (GGGGS)₄ (SEQ ID NO: 120), and (GGGGS)sG (SEQ ID NO: 121).

[0150] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention specifically binds a marker of immune cells. Examples of markers of immune cells include, without limitation, CD25, CD160-TM, PD-1, PDL1, CCR8 and CTLA4.

[0151] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention specifically binds a marker of immune cells selected from the group comprising or consisting of CD25, CD160-TM, PD-1, PDL1, CCR8 and CTLA4.

[0152] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is polyclonal. [0153] In another embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is monoclonal.

[0154] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is selected from the group comprising or consisting of a whole antibody, a single chain antibody, a dimeric single chain antibody, a Fv, a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a defucosylated antibody, a bispecific antibody, a diabody, a triabody, a tetrabody, a nanobody, a domain antibody, and a unibody.

[0155] Antigen-binding fragments of antibodies can be obtained using standard methods. For instance, Fab or F(ab')2 fragments may be produced by protease digestion of the isolated antibodies, according to conventional techniques.

[0156] It will also be appreciated that antibodies or antigen-binding fragments thereof comprised in a fusion protein according to the present invention can be modified using known methods. For example, to slow clearance in vivo and obtain a more desirable pharmacokinetic profile, the antibody or antigen-binding fragment thereof may be modified with polyethylene glycol (PEG). Methods for coupling and site-specifically conjugating PEG to an antibody or antigen-binding fragment thereof are described in, e.g., Leong et al., 2001. Cytokine. 16(3):106-19; Delgado et al., 1996. Br J Cancer. 73(2): 175-82.

[0157] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is a humanized antibody or fragment thereof.

[0158] A “humanized antibody”, as used herein, refers to a chimeric antibody or antigen-binding fragment thereof which contains minimal sequence derived from a nonhuman immunoglobulin. It includes antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell, e.g., by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies or antigen-binding fragments thereof comprised in a fusion protein according to the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody” also includes antibodies and antigenbinding fragment thereof in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In other words, the term “humanized antibody” refers to an antibody or antigen-binding fragment thereof in which the CDRs of a recipient human antibody are replaced by CDRs from a donor non-human antibody. Humanized antibodies or antigenbinding fragments thereof may also comprise residues of donor origin in the framework sequences. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies or antigen-binding fragments thereof may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Humanization can be performed using methods known in the art (e.g, Jones et al., 1986. Nature. 321(6069) :522-5 ; Riechmann et al., 1988. Nature. 332(6162) :323-7 ; Verhoeyen etal., 1988. Science. 239(4847) : 1534-6 ; Presta, 1992. Curr OpinBiotechnol. 3(4):394-8; Patent US4,816,567), including techniques such as “superhumanizing” antibodies (e.g., Tan et al., 2002. J Immunol. 169(2): 1119-25) and “resurfacing” (e.g., Staelens et al., 2006. Mol Immunol. 43(8): 1243-57; Roguska et al., 1994. Proc Natl Acad Sci USA. 91(3):969-73).

[0159] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is a fully or substantially human antibody or fragment thereof.

[0160] The term “substantially human”, in the context of the constant region of a humanized or chimeric antibody or antigen-binding fragment thereof, refers to an amino acid sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more with a human constant region.

[0161] The term “human amino acid sequence”, in this context, refers to an amino acid sequence which is encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes. The present invention also contemplates proteins comprising constant domains of “human” sequence which have been altered, by one or more amino acid additions, deletions or substitutions with respect to the human sequence, excepting those embodiments where the presence of a “fully human hinge region” is expressly required.

[0162] The presence of a “fully human hinge region” in the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention may be beneficial both to minimize immunogenicity and to optimize stability of the antibody. It is considered that one or more amino acid substitutions, insertions or deletions may be made within the constant region of the heavy and/or the light chain, particularly within the Fc region. Amino acid substitutions may result in replacement of the substituted amino acid with a different naturally occurring amino acid, or with a nonnatural or modified amino acid. Other structural modifications are also permitted, such as for example changes in glycosylation pattern (e.g., by addition or deletion of N- or O- linked glycosylation sites).

[0163] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is an engineered antibody or fragment thereof.

[0164] Engineered antibodies include those in which modifications have been made to framework residues within VH and/or VL, e.g., to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “back-mutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “back-mutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such “back- mutated” antibodies are also intended to be encompassed by the invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell-epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0165] Engineered antibodies comprised in a fusion protein according to the present invention include those in which modifications have been made to modify the glycosylation.

[0166] For example, the at least one antibody comprised in a fusion protein according to the invention is aglycosyled (z.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen or alter the ADCC activity of the antibody. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al (incorporated herein by reference). Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered fucosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. For example, EPl 176195 (incorporated herein by reference) describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the at least one antibody or antigen- binding fragment thereof comprised in a fusion protein according to the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransf erase. PCT Publication WO 03/035835 (incorporated herein by reference) describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 (incorporated herein by reference) describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (.eurekainc.com/a&boutus/companyoverview.html). Alternatively, the at least one antibody (preferably the at least one monoclonal antibody) comprised in a fusion protein according to the present invention can be produced in yeasts or filamentous fungi engineered for mammalian-like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).

[0167] Engineered antibodies comprised in a fusion protein according to the present invention include those in which modification have been made to promote heterodimerization of the heavy chains.

[0168] For example, the antibodies comprised in the fusion protein according to the present invention may be modified by knobs-into-holes (KIHs) technology to promote heterodimerization of the heavy chains of the antibody, enabling thereof the formation of the fusion protein according to the present invention by heterodimerization. As used herein, KIH is a method well-known by the skilled artisan in the art, consisting of engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization. [0169] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is from the IgG class.

[0170] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is from the human IgGl subclass. In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is thus an IgGl antibody, preferably a human IgGl antibody.

[0171] In another embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is from the human IgG2 subclass.

[0172] The Fc region of IgG antibodies interacts with cellular Fey receptors (FcyR) to stimulate and regulate downstream effector mechanisms. There are five activating receptors, namely FcyRI (CD64), FcyRIIa (CD32a), FcyRIIc (CD32c), FcyRIIIa (CD 16a) and FcyRIIIb (CD16b), and one inhibitory receptor FcyRIIb (CD32b). The communication of IgG antibodies with the immune system is controlled and mediated by FcyRs, which relay the information sensed and gathered by antibodies to the immune system, providing a link between the innate and adaptive immune systems, and particularly in the context of biotherapeutics (Hayes J et al., 2016. J Inflamm Res 9: 209- 219).

[0173] IgG subclasses vary in their ability to bind to FcyR and this differential binding determines their ability to elicit a range of functional responses. For example, in humans, FcyRIIIa is the major receptor involved in the activation of antibody-dependent cell- mediated cytotoxicity (ADCC) and IgG3 (followed closely by IgGl) display the highest affinities for this receptor, reflecting their ability to potently induce ADCC. IgG2 have been shown to have weak binding for this receptor.

[0174] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention binds FcyR with high affinity, preferably binds an activating receptor with high affinity. [0175] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention binds FcyRI and/or FcyRIIa and/or FcyRIIc and/or FcyRIIIa and/or FcyRIIIb with high affinity.

[0176] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is an IgGl antibody (preferably a human IgGl antibody) or a fragment thereof, and binds to at least one Fc activating receptor. For example, the at least one antibody or antigen- binding fragment thereof may bind to one or more receptor selected from FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa and FcyRIIIb. In one embodiment, the at least one antibody or antigen-binding fragment thereof is capable of binding to FcyRIIIa. In one embodiment, the at least one antibody or antigen-binding fragment thereof is capable of binding to FcyRIIa. In one embodiment, the at least one antibody or antigen-binding fragment thereof is capable of binding to FcyRIIIa, FcyRIIc and optionally FcyRI. In one embodiment, the at least one antibody or antigen-binding fragment thereof is capable of binding to FcyRIIIa, FcyRIIa and optionally FcyRI.

[0177] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention binds to at least one activating Fey receptor with a dissociation constant of less than about 10'⁶M, 10'⁷M, 10" ⁸M, 10'⁹M or 10'¹⁰M.

[0178] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is an IgGl antibody (preferably a human IgGl antibody) or a fragment thereof and binds to FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and/or FcyRIIIb with a higher affinity than it binds to FcyRIIb, with low affinity.

[0179] In one embodiment, the fusion protein comprises one, two or more antibody(ies) or antigen-binding fragment(s) thereof.

[0180] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention specifically binds CD25. [0181] Thus, in one embodiment, the fusion protein according to the present invention comprises: i. at least one antibody or antigen-binding fragment thereof, which specifically binds to CD25, ii. at least one cytokine, preferably IL-15, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, and iv. at least one cleavable linker, wherein the at least one cleavable linker links the at least one cytokine binding moiety to any one of the elements of the fusion protein.

[0182] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention may comprise human heavy chain constant regions sequences and allow to target, block, and/or deplete CD25- expressing cells to which they are bound.

[0183] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention depletes CD25- expressing cells to which it is bound. In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention depletes Tregs to which it is bound. In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention also depletes or reduces tumor infiltrating regulatory T cells to which it is bound.

[0184] The term “deplete” or “depleting”, with respect to CD25-expressing cells or Tregs refers to the killing, elimination, lysis or induction of such killing, elimination or lysis, so as to negatively affect the number or proportion of CD25 expressing cells present in a sample or in a subject. In one embodiment, the at least one antibody or antigen binding fragment thereof comprised in a fusion protein according to the present invention allows targeting, blocking proliferation, and/or depleting CD25-expressing cells or Treg cells. In one embodiment, the depletion is via ADCC. In one embodiment, the depletion is via ADCP. In one embodiment, the depletion is via CDC. [0185] Thus, in one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention leads, directly or indirectly, to the depletion of CD25-expressing cells (e.g., leads to a 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85% or greater elimination or decrease in number of CD25 expressing cells).

[0186] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention does not inhibit the binding of interleukin-2 (IL-2) to CD25 and depletes Tregs to which it is bound.

[0187] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention induces antibody dependent cellular cytotoxicity (ADCC).

[0188] The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated cytotoxicity induced in an antibody-dependent manner when the Fc region of said antibody bound to its antigen binds to the Fc receptor on effector cells such as natural killer cells, macrophages, neutrophils, eosinophils and mononuclear cells (e.g., peripheral blood mononuclear cells), thereby leading to lysis of the target cell. ADCC can be measured using assays that are known and available in the art (e.g., Clynes etal. (1998) Proc Natl Acad Sci USA 95, 652-6).

[0189] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is from the IgGl (preferably human IgGl) subclass and has ADCC activity.

[0190] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention induces antibodydependent cell-mediated phagocytosis (ADCP).

[0191] The term “antibody-dependent cell-mediated phagocytosis” (ADCP) or “opsonisation” refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., phagocytes, macrophages) that express Fc receptors (FcRs) recognize antibody bound on a target cell and induce phagocytosis of the target cell. ADCP can be measured using assays that are known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6).

[0192] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is from the IgGl (preferably human IgGl) subclass and has ADCP activity.

[0193] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention induces complementdependent cytotoxicity (CDC).

[0194] The term “complement-dependent cytotoxicity” (CDC) refers to the induction of the lysis of antigen-expressing cells recognized by an antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) complexed with a cognate antigen. CDC can be measured using assays that are known and available in the art (e.g., Clynes et al. (1998) Proc Natl Acad Sci USA 95, 652-6 ; Gazzano- Santaro et al., J. Immunol. Methods, 202: 163 (1996)).

[0195] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention is from the IgGl (preferably human IgGl) subclass and has CDC activity.

[0196] The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity and phagocytosis. Thus, as discussed herein, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity/phagocytosis.

[0197] In one embodiment, the at least one antibody or antigen-binding fragment thereof according to the present invention lacks an Fc domain (e.g., lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype (preferably of human IgG2 or IgG4). [0198] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention does not comprise an Fc region that mediates ADCC, ADCP and/or CDC.

[0199] In one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention does not induce ADCC, ADCP and/or CDC.

[0200] Thus, in one embodiment, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention does not induce, directly or indirectly, the depletion of CD25- expressing cells (e.g., do not induce a 10%, 20%, 50%, 60% or greater elimination or decrease in number of CD25 cells). For example, the at least one antibody or antigen-binding fragment thereof comprised in a fusion protein according to the present invention does not comprise an Fc domain capable of substantially binding to an FcyRIIIA (CD16) polypeptide.

[0201] Examples of antibodies and fragments thereof that specifically binds CD25 include, without limitation, the antibodies described in the international patent publications WO2020/234399, WO2022/106663 and WO 2022/106665.

[0202] In the following, and unless explicitly mentioned otherwise, CDR numbering and definitions are according to the IMGT® numbering system.

[0203] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: YISYDGDNTYYRDSVKG (SEQ ID NO: 2); and

- VH-CDR3 : GGNSGYD (SEQ ID NO : 3);

- VL-CDR1 : RASQNVNKFLN (SEQ ID NO: 4);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6), or any CDR having at least 70% of identity with SEQ ID NOs: 1-6. [0204] Example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 2 and 3 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 4, 5 and 6 are MAB2, MAB3, MAB6 and MAB7.

[0205] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 40 and a VL comprising or consisting of the sequence SEQ ID NO: 41, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 40-41. An example of an antibody comprising a VH and a VL with SEQ ID NO: 40 and 41, respectively, is MAB2.

SEQ ID NO: 40

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPKKGLEWVAYIS YDGDNTYYRDSVKGRFTISRDNAQSTLYLQMNSLRAEDTAVYYCTTGGNSGY DWGQGTLVTVSS

SEQ ID NO: 41

DIQMTQSPSSLSASVGDRVTITCRASQNVNKFLNWYQQKPGKAPRRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0206] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 40 and a VL comprising or consisting of the sequence SEQ ID NO: 42, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 40, 42. An example of an antibody comprising a VH and a VL with SEQ ID NO: 40 and 42, respectively, is MAB3.

SEQ ID NO: 42

DIQMTQSPSSLSASVGDRVTITCRASQNVNKFLNWYQQKPGKAPRRLIYGTNSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0207] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 43 and a VL comprising or consisting of the sequence SEQ ID NO: 41, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 43, 41. An example of an antibody comprising a VH and a VL with SEQ ID NO: 43 and 41, respectively, is MAB6.

SEQ ID NO: 43

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAYIS YDGDNTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCTTGGNSGY DWGQGTLVTVSS

[0208] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 43 and a VL comprising or consisting of the sequence SEQ ID NO: 42, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 43, 42. An example of an antibody comprising a VH and a VL with SEQ ID NO: 43 and 42, respectively, is MAB7.

[0209] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: YISYDGDNTYYADSVKG (SEQ ID NO: 7); and

- VH-CDR3 : GGNSGYD (SEQ ID NO : 3);

- VL-CDR1 : RASQNVNKFLN (SEQ ID NO: 4);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6), or any CDR having at least 70% of identity with SEQ ID NOs: 1, 7, 3, 4-6.

[0210] Example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 7 and 3 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 4, 5 and 6 are MAB 10 and MAB 11.

[0211] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 44 and a VL comprising or consisting of the sequence SEQ ID NO: 41, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 44, 41. An example of an antibody comprising a VH and a VL with SEQ ID NO: 44 and 41, respectively, is MAB10.

SEQ ID NO: 44

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAYIS YDGDNTYYADSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCTTGGNSGY DWGQGTLVTVSS

[0212] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 44 and a VL comprising or consisting of the sequence SEQ ID NO: 42 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 44, 42. An example of an antibody comprising a VH and a VL with SEQ ID NO: 44 and 42, respectively, is MAB11.

[0213] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1)

- VH-CDR2: YISYDGDNTYYADSVKG (SEQ ID NO: 7); and

- VH-CDR3 : GGNSGYD (SEQ ID NO : 3);

- VL-CDR1 : KGSQNVNKFLN (SEQ ID NO: 8);

- VL-CDR2: GTNSLQT (SEQ ID NO: 9); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6).

Or any CDR having at least 70% of identity with SEQ ID NOs: 1, 7, 3, 8, 9, 6.

[0214] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 7 and 3 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 8, 9 and 6 is MAB9.

[0215] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 44 and a VL comprising or consisting of the sequence SEQ ID NO: 45 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 44, 45. An example of an antibody comprising a VH and a VL with SEQ ID NO: 44 and 45, respectively, is MAB9.

SEQ ID NO: 45

DIQMTQSPSSLSASVGDRVTITCKGSQNVNKFLNWYQQKLGEAPRRLIYGTNSL QTGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0216] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : SHAMA (SEQ ID NO: 10);

- VH-CDR2: YISYDGDNTYYADSVKG (SEQ ID NO: 7); and

- VH-CDR3 : GGNSGYD (SEQ ID NO: 3)

- VL-CDR1 : KGSQNVNKFLN (SEQ ID NO: 8);

- VL-CDR2: GTNSLQT (SEQ ID NO: 9); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6).

Or any CDR having at least 70% of identity with SEQ ID NOs: 10, 7, 3, 8, 9 or 6.

[0217] Examples of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 10, 7 and 3 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 8, 9 and 6 are MAB13 and MAB17.

[0218] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 46 and a VL comprising or consisting of the sequence SEQ ID NO: 45, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 46, 45. An example of an antibody comprising a VH and a VL with SEQ ID NO: 46 and 45, respectively, is MAB13.

SEQ ID NO: 46

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHAMAWVRQAPGKGLEWVAYIS YDGDNTYYADSVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCTTGGNSGY DWGQGTLVTVSS [0219] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 47 and a VL comprising or consisting of the sequence SEQ ID NO: 45, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 47, 45. An example of an antibody comprising a VH and a VL with SEQ ID NO: 47 and 45, respectively, is MAB17.

SEQ ID NO: 47

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHAMAWVRQAPGKGLEWVAYIS YDGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGGNSGY DWGQGTLVTVSS

[0220] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : SHAMA (SEQ ID NO: 10);

- VH-CDR2: YISYDGDNTYYADSVKG (SEQ ID NO: 7); and

- VH-CDR3 : GGNSGYD (SEQ ID NO: 3),

- VL-CDR1 : RASQNVNKFLN (SEQ ID NO: 4);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6).

Or any CDR having at least 70% of identity with SEQ ID NOs: 10, 7, 3, 4-6.

[0221] Examples of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 10, 7 and 3 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 4, 5 and 6 are MAB14, MAB15, MAB18 and MAB19.

[0222] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 46 and a VL comprising or consisting of the sequence SEQ ID NO: 41, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 46, 41. An example of an antibody comprising a VH and a VL with SEQ ID NO: 46 and 41, respectively, is MAB14. [0223] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 46 and a VL comprising or consisting of the sequence SEQ ID NO: 42, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 46, 42. An example of an antibody comprising a VH and a VL with SEQ ID NO: 46 and 42, respectively, is MAB15.

[0224] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 47 and a VL comprising or consisting of the sequence SEQ ID NO: 41, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 47, 41. An example of an antibody comprising a VH and a VL with SEQ ID NO: 47 and 41, respectively, is MAB18.

[0225] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 47 and a VL comprising or consisting of the sequence SEQ ID NO: 42, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 47, 42. An example of an antibody comprising a VH and a VL with SEQ ID NO: 47 and 42, respectively, is MAB19.

[0226] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GWNSGYD (SEQ ID NO: 12);

- VL-CDR1 : RASQSVNSFLN (SEQ ID NO: 13);

- VL-CDR2: GTSSLQS (SEQ ID NO: 14); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 12, 13, 14 or 6. [0227] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 12 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 13, 14 and 6 is H07.

[0228] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 48 and a VL comprising or consisting of the sequence SEQ ID NO: 49, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 48, 49. An example of an antibody comprising a VH and a VL with SEQ ID NO: 48 and 49, respectively, is H07.

SEQ ID NO: 48

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTTGWNSGY DWGQGTLVTVSS

SEQ ID NO: 49

DIQMTQSPSSLSASVGDRVTITCRASQSVNSFLNWYQQKPGKAPRRLIYGTSSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0229] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GWNSGYD (SEQ ID NO: 12);

- VL-CDR1 : RASQSVNSYLN (SEQ ID NO: 15);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 12, 15, 5 or 16.

[0230] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 12 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 15, 5 and 16 is G02. [0231] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 48 and a VL comprising or consisting of the sequence SEQ ID NO: 50, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 48, 50. An example of an antibody comprising a VH and a VL with SEQ ID NO: 48 and 50, respectively, is G02.

SEQ ID NO: 50

DIQMTQSPSSLSASVGDRVTITCRASQSVNSYLNWYQQKPGKAPRRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0232] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GWNSGYD (SEQ ID NO: 12);

- VL-CDR1 : RASQSISSYLN (SEQ ID NO: 17);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 12, 17, 5 or 16.

[0233] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 12 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 17, 5 and 16 is E04.

[0234] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 48 and a VL comprising or consisting of the sequence SEQ ID NO: 51, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 48, 51. An example of an antibody comprising a VH and a VL with SEQ ID NO: 48 and 51, respectively, is E04.

SEQ ID NO: 51 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPRRLIYGTNSLQ SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0235] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GWNSGYD (SEQ ID NO: 12);

- VL-CDR1: RASQSVSSFLN (SEQ ID NO: 18);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6).

Or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 12, 18, 5 or 6.

[0236] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 12 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 18, 5 and 6 is D05.

[0237] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 52 and a VL comprising or consisting of the sequence SEQ ID NO: 53, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 52, 53. An example of an antibody comprising a VH and a VL with SEQ ID NO: 52 and 53, respectively, is D05.

SEQ ID NO: 52

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGWNSGY DWGQGTLVTVSS

SEQ ID NO: 53

DIQMTQSPSSLSASVGDRVTITCRASQSVSSFLNWYQQKPGEAPRRLIYGTNSLQ

SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK [0238] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSVSKFLN (SEQ ID NO: 20);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 20, 5 or 16.

[0239] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 20, 5 and 16 is H09.

[0240] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 54 and a VL comprising or consisting of the sequence SEQ ID NO: 55, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 54, 55. An example of an antibody comprising a VH and a VL with SEQ ID NO: 54 and 55, respectively, is H09.

SEQ ID NO: 54

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 55

DIQMTQSPSSLSASVGDRVTITCRASQSVSKFLNWYQQKPGKAPRRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0241] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL): - VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQNINSFLN (SEQ ID NO: 21);

- VL-CDR2: GTSSLQS (SEQ ID NO: 14); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 21, 14 or 16.

[0242] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 21, 14 and 16 is E04-2.

[0243] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 56 and a VL comprising or consisting of the sequence SEQ ID NO: 57, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 57. An example of an antibody comprising a VH and a VL with SEQ ID NO: 56 and 57, respectively, is E04-2.

SEQ ID NO: 56

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 57

DIQMTQSPSSLSASVGDRVTITCRASQNINSFLNWYQQKPGKAPRRLIYGTSSLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0244] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19); - VL-CDR1 : RASQNISSFLN (SEQ ID NO: 22);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 22, 5 or 16.

[0245] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 22, 5 and 16 is BO 1.

[0246] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 58 and a VL comprising or consisting of the sequence SEQ ID NO: 59 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 58, 59. An example of an antibody comprising a VH and a VL with SEQ ID NO: 58 and 59, respectively, is B01.

SEQ ID NO: 58

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 59

DIQMTQSPSSLSASVGDRVTITCRASQNISSFLNWYQQKPGEAPRRLIYGTNSLQ SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0247] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSISSFLN (SEQ ID NO: 23);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 23, 5 or 6.

[0248] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 23, 5 and 6 is C01.

[0249] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 54 and a VL comprising or consisting of the sequence SEQ ID NO: 60, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 54, 60. An example of an antibody comprising a VH and a VL with SEQ ID NO: 54 and 60, respectively, is COL

SEQ ID NO: 60

DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGEAPRRLIYGTNSLQ SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0250] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSVSSFLN (SEQ ID NO: 18);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 18, 5 or 16.

[0251] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 18, 5 and 16 is GO 1.

[0252] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 54 and a VL comprising or consisting of the sequence SEQ ID NO: 61, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 54, 61. An example of an antibody comprising a VH and a VL with SEQ ID NO: 54 and 61, respectively, is G01.

SEQ ID NO: 61

DIQMTQSPSSLSASVGDRVTITCRASQSVSSFLNWYQQKPGKAPKRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0253] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQNVSSFLN (SEQ ID NO: 24);

- VL-CDR2: GTSSLQS (SEQ ID NO: 14); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 24, 14 or 6.

[0254] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 24, 14 and 6 is HOL

[0255] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 54 and a VL comprising or consisting of the sequence SEQ ID NO: 62, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 54, 62. An example of an antibody comprising a VH and a VL with SEQ ID NO: 54 and 62, respectively, is HOL

SEQ ID NO: 62

DIQMTQSPSSLSASVGDRVTITCRASQNVSSFLNWYQQKPGKAPRRLIYGTSSL

QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK [0256] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSINSFLN (SEQ ID NO: 25);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 25, 5 or 16.

[0257] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 25, 5 and 16 is G02-2.

[0258] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 56 and a VL comprising or consisting of the sequence SEQ ID NO: 63, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 63. An example of an antibody comprising a VH and a VL with SEQ ID NO: 56 and 63, respectively, is G02-2.

SEQ ID NO: 63

DIQMTQSPSSLSASVGDRVTITCRASQSINSFLNWYQQKPGKAPRRLIYGTNSLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0259] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQNINSFLN (SEQ ID NO: 21); - VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 21, 5 or 6.

[0260] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 21, 5 and 6 is B07.

[0261] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 54 and a VL comprising or consisting of the sequence SEQ ID NO: 64, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 54, 64. An example of an antibody comprising a VH and a VL with SEQ ID NO: 54 and 64, respectively, is B07.

SEQ ID NO: 64

DIQMTQSPSSLSASVGDRVTITCRASQNINSFLNWYQQKPGEAPRRLIYGTNSLQ SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0262] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYADSVKG (SEQ ID NO: 11); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSVSSYLN (SEQ ID NO: 26);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6).

Or any CDR having at least 70% of identity with SEQ ID NOs: 1, 11, 19, 26, 5 or 6.

[0263] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 11 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 26, 5 and 6 is H08. [0264] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 65 and a VL comprising or consisting of the sequence SEQ ID NO: 66, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 65, 66. An example of an antibody comprising a VH and a VL with SEQ ID NO: 65 and 66, respectively, is H08.

SEQ ID NO: 65

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 66

DIQMTQSPSSLSASVGDRVTITCRASQSVSSYLNWYQQKPGKAPRRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0265] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYRDSVKG (SEQ ID NO: 27); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSISSFLN (SEQ ID NO: 23);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 27, 19, 23, 5 or 16.

[0266] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 27 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 23, 5 and 16 is D01.

[0267] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 67 and a VL comprising or consisting of the sequence SEQ ID NO: 68, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 67, 68. An example of an antibody comprising a VH and a VL with SEQ ID NO: 67 and 68, respectively, is DOI.

SEQ ID NO: 67

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYRDSVKGRFTISRDNAQSTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 68

DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAPRRLIYGTNSLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0268] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYRDSVKG (SEQ ID NO: 27); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQNINSFLN (SEQ ID NO: 21);

- VL-CDR2: GTSSLQS (SEQ ID NO: 14); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16); or any CDR having at least 70% of identity with SEQ ID NOs: 1, 27, 19, 21, 14 or 16.

[0269] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 27 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 21, 14 and 16 is B05.

[0270] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 69 and a VL comprising or consisting of the sequence SEQ ID NO: 70, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 69, 70. An example of an antibody comprising a VH and a VL with SEQ ID NO: 69 and 70, respectively, is B05. SEQ ID NO: 69

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 70

DIQMTQSPSSLSASVGDRVTITCRASQNINSFLNWYQQKPGKAPRRLIYGTSSLQ SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0271] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNKYYRDSVKG (SEQ ID NO: 27); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQNVSSFLN (SEQ ID NO: 24);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16).

Or any CDR having at least 70% of identity with SEQ ID NOs: 1, 27, 19, 24, 5 or 16.

[0272] Examples of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 27 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 24, 5 and 16 are G09 et H02.

[0273] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 71 and a VL comprising or consisting of the sequence SEQ ID NO: 72, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 71, 72. An example of an antibody comprising a VH and a VL with SEQ ID NO: 71 and 72, respectively, is G09. SEQ ID NO: 71

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYRDSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 72

DIQMTQSPSSLSASVGDRVTITCRASQNVSSFLNWYQQKPGEAPRRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0274] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 73 and a VL comprising or consisting of the sequence SEQ ID NO: 72, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 73, 72. An example of an antibody comprising a VH and a VL with SEQ ID NO: 73 and 72, respectively, is H02.

SEQ ID NO: 73

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNKYYRDSVKGRFTISRDNSQSTLYLQMNSLRAEDTAVYYCTTGLNSGYD WGQGTLVTVSS

[0275] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGSNTYYADSVKG (SEQ ID NO: 28); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQNVSSFLN (SEQ ID NO: 24);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYNSWPWT (SEQ ID NO: 16).

Or any CDR having at least 70% of identity with SEQ ID NOs: 1, 28, 19, 24, 5 or 16. [0276] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 28 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 24, 5 and 16 is F03.

[0277] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 74 and a VL comprising or consisting of the sequence SEQ ID NO: 75, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 74, 75. An example of an antibody comprising a VH and a VL with SEQ ID NO: 74 and 75, respectively, is F03.

SEQ ID NO: 74

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGSNTYYADSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 75

DIQMTQSPSSLSASVGDRVTITCRASQNVSSFLNWYQQKPGKAPRRLIYGTNSL QSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNSWPWTFGQGTKLEIK

[0278] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises the following complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDR1 : NHAMA (SEQ ID NO: 1);

- VH-CDR2: VISYDGDNKYYADSVKG (SEQ ID NO: 29); and

- VH-CDR3 : GLNSGYD (SEQ ID NO: 19);

- VL-CDR1 : RASQSINSFLN (SEQ ID NO: 25);

- VL-CDR2: GTNSLQS (SEQ ID NO: 5); and

- VL-CDR3 : QQYTSWPWT (SEQ ID NO: 6).

Or any CDR having at least 70% of identity with SEQ ID NOs: 1, 29, 19, 25, 5 or 16.

[0279] An example of antibodies comprising a heavy chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 1, 29 and 19 and a light chain comprising CDR1, CDR2 and CDR3 with SEQ ID NOs 25, 5 and 6 is B12. [0280] In one embodiment, the anti-CD25 antibody or antigen-binding fragment comprises a VH comprising or consisting of the sequence SEQ ID NO: 76 and a VL comprising or consisting of the sequence SEQ ID NO: 77, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 76, 77. An example of an antibody comprising a VH and a VL with SEQ ID NO: 76 and 77, respectively, is B12.

SEQ ID NO: 76

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMAWVRQAPGKGLEWVAVIS YDGDNKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTTGLNSGY DWGQGTLVTVSS

SEQ ID NO: 77

DIQMTQSPSSLSASVGDRVTITCRASQSINSFLNWYQQKPGEAPRRLIYGTNSLQ SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYTSWPWTFGQGTKLEIK

[0281] The present invention further relates to fusion proteins comprising anti-CD25 antibodies, as defined hereinbelow. It will be understood by the skilled artisan in the art that the anti-CD25 antibodies comprised in any one of the fusion proteins defined hereinbelow may comprise any one of the combinations of CDRs as defined hereinabove and/or any one of the combinations of VH and VL as defined hereinabove.

[0282] The present invention further relates to a fusion protein comprising: i. an antibody or a fragment thereof that specifically binds CD25, as defined hereinabove, ii. a human IL-15 or a variant or fragment thereof, as defined hereinabove, iii. a cytokine binding moiety capable of binding the human IL-15 or a variant or fragment thereof, as defined hereinabove, wherein the human IL-15 or a variant or fragment thereof is linked to the C-terminal of one heavy chain of the antibody with an uncleavable linker as defined hereinabove, wherein the cytokine binding moiety is linked to the C-terminal of the other heavy chain of the antibody with a cleavable linker as defined hereinabove.

[0283] An example of such fusion protein is provided in Figure 21A. [0284] In one embodiment, the cleavage of the cleavable linker releases the cytokine binding moiety, thereby leading to the unmasking of the IL-15.

[0285] In one embodiment, the human IL- 15 comprises or consists of the amino acid sequence SEQ ID NO: 31, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 31.

[0286] In one embodiment, the cytokine binding moiety comprises or consists of at least one chain of the human IL- 15 receptor, or a fragment or a variant thereof.

[0287] In one embodiment, the cytokine binding moiety comprises or consists of the P chain of the human IL- 15 receptor, or a variant or fragment thereof. In one embodiment, the cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 37, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 37.

[0288] In one embodiment, the cytokine binding moiety comprises or consists of the domain DI of the P chain of the human IL- 15 receptor, or a fragment or variant thereof. In one embodiment, the at least one cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 38 or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 38.

[0289] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PX(GGGGS)_q]t wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3, and wherein X is at least one protease recognition site. In one embodiment, said cleavable linker may further comprise one or several amino acid residues, such as a serine residue, after the at least one protease recognition site.

[0290] In one embodiment, the at least one linker is a linker glycine-serine polymer, comprising PLGLA (SEQ ID NO: 102) and/or PLGL (SEQ ID NO: 127).

[0291] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PPLGLAGFLGS(GGGGS)_q]t wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3. [0292] In one embodiment, the cleavable linker is a linker comprising or consisting of GGGGSPLGLAGFLGS (SEQ ID NO: 30).

[0293] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PPLGLA(GGGGS)_q]t (SEQ ID NO: 125), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3.

[0294] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence GGGGSPLGLAGGGGS (SEQ ID NO: 129).

[0295] In one embodiment, the uncleavable linker is a glycine-serine polymer.

[0296] In one embodiment, the uncleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t wherein p and q independently range from 0 to 8, wherein t ranges from 1 to 3.

[0297] In one embodiment, the uncleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t wherein p ranges from 1 to 6, q ranges from 0 to 4, wherein t ranges from 1 to 3.

[0298] In one embodiment, the uncleavable linker is a linker comprising or consisting of (GGGGS)₂GGGG (SEQ ID NO: 116).

[0299] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, as defined hereinabove,

From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, as defined hereinabove, and Two light chains of an anti-CD25 antibody, as defined hereinabove.

[0300] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements: One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and the sequence SEQ ID NO: 78, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 78, One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one of the combinations of VH-CDRs or one of the VH as defined hereinabove and any one of the sequences SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 128, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 79-80 or 128,

Two light chains of an anti-CD25 antibody, comprising or consisting of one of the combinations of VL-CDRs or one of the VL as defined hereinabove, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 81.

[0301] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and the sequence SEQ ID NO: 78, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 78,

One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with a sequence SEQ ID NO: 56 and either the sequence SEQ ID NO: 79 or SEQ ID NO: 80, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 79-80,

Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 63, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 63, 81. [0302] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain comprising or consisting of the sequence SEQ ID NO: 144 or SEQ ID NO: 147, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 144 or 147,

One heavy chain comprising or consisting of the sequence SEQ ID NO: 145, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 145,

Two light chains with SEQ ID NO: 146, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 145.

[0303] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 44, and the sequence SEQ ID NO: 78, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 44, 78,

One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with a sequence SEQ ID NO: 44 and the sequence SEQ ID NO: 128, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 44, 128,

Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 41, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 41, 81.

[0304] An example of a fusion protein as defined hereinabove comprises or consists of the following elements: One heavy chain comprising or consisting of the sequence SEQ ID NO: 133 or SEQ ID NO: 135, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 133 or 135,

One heavy chain comprising or consisting of the sequence SEQ ID NO: 134, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 134,

Two light chains with SEQ ID NO: 132, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 132.

[0305] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with a sequence SEQ ID NO: 56 and the sequence SEQ ID NO: 128, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 128,

Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 57, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 57, 81.

[0306] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain comprising or consisting of the sequence SEQ ID NO: 136 or SEQ ID NO: 139, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 136 or 139, One heavy chain comprising or consisting of the sequence SEQ ID NO: 137, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 137,

Two light chains with SEQ ID NO: 138, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 138.

[0307] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 69, and the sequence SEQ ID NO: 78, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 69, 78,

One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with a sequence SEQ ID NO: 69 and the sequence SEQ ID NO: 128, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 69, 128,

Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 70, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 70, 81.

[0308] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain comprising or consisting of the sequence SEQ ID NO: 140 or SEQ ID NO: 143, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 140 or 143,

On heavy chain comprising or consisting of the sequence SEQ ID NO: 141, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 141, Two light chains with SEQ ID NO: 142, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 142.

[0309] In one embodiment, the fusion protein as described hereinabove further comprises an IL- 15 receptor a sushi domain, or a variant or fragment thereof. In one embodiment, the IL-15 receptor a sushi domain, or a variant or fragment thereof, is linked to the human IL- 15, or a variant or fragment thereof, with an uncleavable linker as defined hereinabove.

[0310] Thus, the present invention further relates to a fusion protein comprising: i. an antibody that specifically binds CD25, as defined hereinabove, ii. a human IL-15 or a variant or fragment thereof, as defined hereinabove, iii. a cytokine binding moiety capable of binding the human IL-15 or a variant or fragment thereof, as defined hereinabove, iv. an IL- 15 receptor a sushi domain or a variant or fragment thereof, as defined hereinabove, wherein the human IL-15 or a variant or fragment thereof is linked to the C-terminal of one heavy chain of the antibody with an uncleavable linker as defined hereinabove, and wherein the IL- 15 receptor a sushi domain or a variant or fragment thereof is linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker as defined hereinabove, wherein the cytokine binding moiety is linked to the C-terminal of the other heavy chain of the antibody with a cleavable linker as defined hereinabove.

[0311] An example of such fusion protein is provided in Figure 21B.

[0312] In one embodiment, the cleavage of the cleavable linker releases the cytokine binding moiety, thereby leading to the unmasking of the IL-15.

[0313] In one embodiment, the IL-15 receptor a sushi domain comprises or consists of the sequence SEQ ID NO: 107, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 107. [0314] In one embodiment, the uncleavable linker linking the IL-15 receptor a sushi domain or a variant or fragment thereof to the human IL- 15 or a variant or fragment thereof is a glycine-serine polymer.

[0315] In one embodiment, the uncleavable linker linking the IL- 15 receptor a sushi domain or a variant or fragment thereof to the human IL- 15 or a variant or fragment thereof is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p and q independently range from 0 to 8, wherein t ranges from 1 to 3.

[0316] In one embodiment, the uncleavable linker linking the IL- 15 receptor a sushi domain or a variant or fragment thereof to the human IL- 15 or a variant or fragment thereof is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p ranges from 1 to 6, q ranges from 0 to 4, wherein t ranges from 1 to 3.

[0317] In one embodiment, the uncleavable linker linking the IL- 15 receptor a sushi domain or a variant or fragment thereof to the human IL- 15 or a variant or fragment thereof is a linker comprising or consisting of (GGGGS)sG (SEQ ID NO: 121).

[0318] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the L-15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, as defined hereinabove,

[0319] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the IL-15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, comprising or consisting of one of the combinations of VH-CDRs or one of the VH as defined hereinabove and the sequence SEQ ID NO: 82, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 82, One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one of the combinations of VH-CDRs or one of the VH as defined hereinabove, and any one of the sequence SEQ ID NO: 79 or SEQ ID NO: 80, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 79-80,

[0320] An example of a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the IL-15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and the sequence SEQ ID NO: 82, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 82,

One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and any one of the sequences SEQ ID NO: 79 or SEQ ID NO: 80, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56; 79-80, and

Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 63, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 63, 81.

[0321] The present invention further relates to a fusion protein comprising: i. an antibody or a fragment thereof that specifically binds CD25, as defined hereinabove, ii. a human IL-15 or a variant or fragment thereof, as defined hereinabove, iii. a cytokine binding moiety capable of binding the human IL-15 or a variant or fragment thereof, as defined hereinabove, wherein the IL- 15 or a variant or fragment thereof is linked to the C-terminal of one heavy chain of the antibody with an uncleavable linker as defined hereinabove, wherein the cytokine binding moiety is linked to IL-15 or a variant or fragment thereof with a cleavable linker as defined hereinabove.

[0322] An example of such fusion protein is provided in Figure 21C.

[0323] In one embodiment, the cleavage of the cleavable linker releases the cytokine binding moiety, thereby leading to the unmasking of the IL-15.

[0324] In one embodiment, the human IL- 15 comprises or consists of the amino acid sequence SEQ ID NO: 31, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 31.

[0325] In one embodiment, the cytokine binding moiety comprises or consists of at least one chain of the human IL- 15 receptor, or a fragment or a variant thereof.

[0326] In one embodiment, the cytokine binding moiety comprises or consists of the P chain of the human IL- 15 receptor, or a variant or fragment thereof. In one embodiment, the cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 37 or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 37.

[0327] In one embodiment, the cytokine binding moiety comprises or consists of the domain DI of the P chain of the human IL- 15 receptor, or a fragment or variant thereof. In one embodiment, the at least one cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 38, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 38.

[0328] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PX(GGGGS)_q]t (SEQ ID NO: 100), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3, and wherein X is at least one protease recognition site. In one embodiment, said cleavable linker may further comprise one or several amino acid residues, such as a serine residue, after the at least one protease recognition site.

[0329] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PPLGLAGFLGS(GGGGS)_q]t (SEQ ID NO: 111), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3.

[0330] In one embodiment, the cleavable linker is a linker comprising or consisting of GGGGSPLGLAGFLGS(GGGGS)i or GGGGSPLGLAGFLGS(GGGGS)₂.

[0331] In one embodiment, the uncleavable linker is a glycine-serine polymer.

[0332] In one embodiment, the uncleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t(SEQ ID NO: 115), wherein p and q independently range from 0 to 8, wherein t ranges from 1 to 3.

[0333] In one embodiment, the uncleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p ranges from 1 to 6, q ranges from 0 to 4, wherein t ranges from 1 to 3.

[0334] In one embodiment, the uncleavable linker is a linker comprising or consisting of (GGGGS)₂GGGG (SEQ ID NO: 116).

[0335] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL- 15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, as defined hereinabove,

One heavy chain of an anti-CD25 antibody, as defined hereinabove.

Two light chains of an anti-CD25 antibody, as defined hereinabove.

[0336] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and any one of the sequences SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 83-86,

One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one the combinations of VH- CDRs or one of the VH as defined hereinabove, and the sequence SEQ ID NO: 87, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 87,

[0337] As an example, a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and any one of the sequences SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 83-86, One heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and the sequence SEQ ID NO: 87, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 87,

[0338] In one embodiment, the fusion protein as described hereinabove further comprises an IL-15 receptor a sushi domain or a variant or fragment thereof. In one embodiment, the IL- 15 receptor a sushi domain or a variant or fragment thereof is linked to the C-terminal of the other heavy chain of the antibody with an uncleavable linker as defined hereinabove.

[0339] Thus, the present invention further relates to a fusion protein comprising: i. an antibody that specifically binds CD25, as defined hereinabove, ii. a human IL-15 or a variant or fragment thereof, as defined hereinabove, iii. a cytokine binding moiety capable of binding the human IL-15, as defined hereinabove, iv. an IL- 15 receptor a sushi domain or a variant or fragment thereof, wherein the IL- 15 or a variant or fragment thereof is linked to the C-terminal of one heavy chain of the antibody with an uncleavable linker, and wherein the cytokine binding moiety is linked to IL- 15 or a variant or fragment thereof with a cleavable linker, wherein the IL- 15 receptor a sushi domain or a variant or fragment thereof is linked to the C-terminal of the other heavy chain of the antibody with an uncleavable linker.

[0340] An example of such fusion protein is provided in Figure 21D. [0341] In one embodiment, the cleavage of the cleavable linker releases the cytokine binding moiety, thereby leading to the unmasking of the IL-15.

[0342] In one embodiment, the IL-15 receptor a sushi domain comprises or consists of the sequence SEQ ID NO: 107, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 107.

[0343] In one embodiment, the uncleavable linker linking the IL-15 receptor a sushi domain or a variant or fragment thereof to the C-terminal of the other heavy chain of the antibody is a glycine-serine polymer.

[0344] In one embodiment, the uncleavable linker linking the IL- 15 receptor a sushi domain or a variant or fragment thereof to the C-terminal of the other heavy chain of the antibody is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p and q independently range from 0 to 8, wherein t ranges from 1 to 3.

[0345] In one embodiment, the uncleavable linker linking the IL- 15 receptor a sushi domain or a variant or fragment thereof to the C-terminal of the other heavy chain of the antibody is a linker comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p ranges from 1 to 6, q ranges from 0 to 4, wherein t ranges from 1 to 3.

[0346] In one embodiment, the uncleavable linker linking the IL- 15 receptor a sushi domain or a variant or fragment thereof to the C-terminal of the other heavy chain of the antibody comprises or consists of (GGGGS)3 (SEQ ID NO: 119) or (GGGGS)4 (SEQ ID NO: 120).

[0347] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the IL-15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, as defined hereinabove, and

Two light chains of an anti-CD25 antibody, as defined hereinabove.

[0348] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and any one of the sequence SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 83-86,

One heavy chain of an anti-CD25 antibody linked to the IL- 15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and the sequence SEQ ID NO: 88 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 88, and

[0349] As an example, a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and any one of the sequence SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 83-86, One heavy chain of an anti-CD25 antibody linked to the IL- 15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and the sequence SEQ ID NO: 88 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 88, and

Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 63, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 81.

[0350] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and any one of the sequence SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 92, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 89-92,

One heavy chain of an anti-CD25 antibody linked to the IL- 15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and the sequence SEQ ID NO: 93 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 93, and Two light chains of an anti-CD25 antibody, comprising or consisting of one of the combinations of VL-CDRs or one of the VL as defined hereinabove, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 81.

[0351] As an example, a fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, wherein said human IL-15 or a variant or fragment thereof is linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and any one of the sequence SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 92, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 56, 89-92, One heavy chain of an anti-CD25 antibody linked to the IL- 15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and the sequence SEQ ID NO: 93 or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 93, and

[0352] The present invention further relates to a fusion protein comprising: i. an antibody or a fragment thereof that specifically binds CD25, preferably wherein said antibody comprises one the combinations of CDRs recited hereinabove, ii. a human IL-15 or a variant or fragment thereof, iii. a cytokine binding moiety capable of binding the human IL-15 or a variant or fragment thereof, iv. an IL- 15 receptor a sushi domain or a variant or fragment thereof, wherein the IL- 15 receptor a sushi domain or a variant or fragment thereof is linked to the C-terminal of one heavy chain of the antibody with an uncleavable linker, and wherein the IL-15 or a variant or fragment thereof is linked to the IL-15 receptor a sushi domain or a variant or fragment thereof with an uncleavable linker, wherein the cytokine binding moiety is linked to the C-terminal of the other heavy chain of the antibody with a cleavable linker.

[0353] An example of such fusion protein is provided in Figure 21E.

[0354] In one embodiment, the cleavage of the cleavable linker releases the cytokine binding moiety, thereby leading to the unmasking of the IL-15.

[0355] In one embodiment, the human IL-15 comprises or consists of the amino acid sequence SEQ ID NO: 31 or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 31.

[0356] In one embodiment, the cytokine binding moiety comprises or consists of at least one chain of the human IL- 15 receptor, or a fragment or a variant thereof.

[0357] In one embodiment, the cytokine binding moiety comprises or consists of the P chain of the human IL- 15 receptor, or a variant or fragment thereof. In one embodiment, the cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 37, or a variant or fragment thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 37.

[0358] In one embodiment, the cytokine binding moiety comprises or consists of the domain DI of the P chain of the human IL- 15 receptor, or a fragment or variant thereof. In one embodiment, the at least one cytokine binding moiety comprises or consists of the sequence SEQ ID NO: 38, or a fragment or a variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 38.

[0359] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PX(GGGGS)_q]t (SEQ ID NO: 100), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3, and wherein X is at least one protease recognition site. In one embodiment, said cleavable linker may further comprise one or several amino acid residues, such as a serine residue, after the at least one protease recognition site.

[0360] In one embodiment, the cleavable linker is a linker comprising or consisting of the sequence [(GGGGS)_PPLGLAGFLGS(GGGGS)_q]t (SEQ ID NO: 111), wherein p and q independently range from 0 to 2, wherein t ranges from 1 to 3.

[0361] In one embodiment, the cleavable linker is a linker comprising or consisting of GGGGSPLGLAGFLGS (SEQ ID NO: 30).

[0362] In one embodiment, the uncleavable linkers are glycine-serine polymers.

[0363] In one embodiment, the uncleavable linkers are linkers comprising or consisting of the sequence [(GGGGS)_PG_q]t(SEQ ID NO: 115), wherein p and q independently range from 0 to 8, wherein t ranges from 1 to 3.

[0364] In one embodiment, the uncleavable linkers are linkers comprising or consisting of the sequence [(GGGGS)_PG_q]t (SEQ ID NO: 115), wherein p ranges from 1 to 6, q ranges from 0 to 4, wherein t ranges from 1 to 3.

[0365] In one embodiment, the uncleavable linker linking the IL-15 receptor a sushi domain or a variant or fragment thereof to the C-terminal of one heavy chain of the antibody is a linker comprising or consisting of (GGGGS)3 (SEQ ID NO: 119).

[0366] In one embodiment, the uncleavable linker linking the human IL-15 or a variant or fragment thereof to the IL- 15 receptor a sushi domain or a variant or fragment thereof is a linker comprising or consisting of (GGGGS)eGG (SEQ ID NO: 118).

[0367] In one embodiment, the IL-15 receptor a sushi domain comprises or consists of the sequence SEQ ID NO: 107, or a fragment or variant thereof sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 107.

[0368] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements: From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the IL-15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, wherein the IL- 15 receptor a sushi domain or a fragment or variant thereof is linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, as defined hereinabove,

[0369] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements:

One heavy chain of an anti-CD25 antibody linked to the IL- 15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, wherein the IL-15 receptor a sushi domain or a fragment or variant thereof is linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and the sequence SEQ ID NO: 94, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 94,

From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of one the combinations of VH-CDRs or one of the VH as defined hereinabove, and any one of the sequence SEQ ID NO: 79 or SEQ ID NO: 80, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 79-80, and

[0370] In one embodiment, the fusion protein as defined hereinabove comprises or consists of the following elements: One heavy chain of an anti-CD25 antibody linked to the IL- 15 receptor a sushi domain or a fragment or variant thereof with an uncleavable linker, wherein the IL-15 receptor a sushi domain or a fragment or variant thereof is linked to the human IL- 15 or a variant or fragment thereof with an uncleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and the sequence SEQ ID NO: 94, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 94, From N- to C-terminal, one heavy chain of an anti-CD25 antibody linked to the cytokine binding moiety with a cleavable linker, comprising or consisting of the VH with sequence SEQ ID NO: 56, and any one of the sequence SEQ ID NO: 79 or SEQ ID NO: 80, or any sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 79-80, and Two light chains of an anti-CD25 antibody, comprising or consisting of the VL with sequence SEQ ID NO: 63, and the sequence SEQ ID NO: 81, or a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NO: 81.

[0371] Another object of the invention is an isolated nucleic acid encoding a fusion protein according to the present invention.

[0372] An “isolated nucleic acid”, as used herein, is intended to refer to a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.

[0373] In one embodiment, the isolated nucleic acid is purified.

[0374] In one embodiment, the isolated nucleic acid is purified to: greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more by weight of nucleic acid as determined by absorbance methods or fluorescence methods (such as, e.g., by measuring the ratio of absorbance at 260 and 280 nm (A260/280)), and most preferably greater than 96%, 97%, 98% or 99% by weight; or homogeneity as shown by agarose gel electrophoresis and using an intercalating agent such as ethidium bromide, SYBR Green, GelGreen or the like.

[0375] In one embodiment, the nucleic acid comprises a sequence encoding at least a heavy chain or a light chain of the antibody or antigen-binding fragment thereof as defined hereinabove. In one embodiment, the nucleic acid comprises a sequence encoding at least a heavy chain and a light chain of the antibody or antigen-binding fragment thereof as defined hereinabove.

[0376] In one embodiment, the nucleic acid comprises a sequence encoding a cytokine as defined hereinabove.

[0377] In one embodiment, the nucleic acid comprises a sequence encoding a cytokine binding moiety as defined hereinabove.

[0378] In one embodiment, the nucleic acid comprises a sequence encoding at least one cleavable linker as defined hereinabove. In one embodiment, the nucleic acid comprises a sequence encoding at least one uncleavable linker as defined hereinabove.

[0379] In one embodiment, the nucleic acid comprises a sequence encoding an IL-15 receptor a sushi domain or a variant or fragment thereof as defined hereinabove.

[0380] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as for example plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0381] Thus, another object of the present invention is an expression vector comprising a nucleic acid encoding the fusion protein according to the present invention.

[0382] The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform a host and promote expression (e.g. transcription and translation) of the introduced sequence. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said fusion protein upon administration to a host. 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 fusion protein 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 replication-defective recombinant viruses may be found in the art.

[0383] In one embodiment, the expression vector according to the present invention is monoci stronic.

[0384] By “monocistronic”, it is meant that a single nucleic acid is expressed in a single expression vector.

[0385] In one embodiment, the expression vector according to the present invention is polycistronic.

[0386] By “polycistronic”, it is meant that at least two or more nucleic acids are expressed in a single expression vector.

[0387] Another object of the invention is an isolated host cell comprising said vector. Said host cell may be used for the recombinant production of the fusion proteins of the present invention. [0388] In an embodiment, host cells may be prokaryote, yeast, or eukaryote cells, preferably mammalian cells, such as, for example: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL 1581 ; ATCC CRL 8287) or NSO (HP A culture collections no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM’s PERC-6 cell line. Expression vectors suitable for use in each of these host cells are also generally known in the art. It should be noted that the term “host cell” generally refers to a cultured cell line. In one embodiment, whole human beings into which an expression vector encoding a fusion protein according to the invention has been introduced are excluded from the definition of a “host cell”.

[0389] Another object of the present invention is a method of producing and purifying the fusion protein as described herein.

[0390] In one embodiment, the method comprises: introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described hereinabove into a competent host cell; culturing in vitro or ex vivo the host cells transformed with the nucleic acid or expression vector, under conditions suitable for expression of the fusion protein; optionally, selecting the cells which express and/or secrete said fusion protein; and recovering the expressed fusion protein.

[0391] This recombinant process can be used for large scale production of fusion proteins, including fusion proteins for in vitro, ex vivo and/or in vivo therapeutic uses.

[0392] In one embodiment, the expressed fusion protein is further purified.

[0393] Methods to purify a protein, in particular a fusion protein as defined hereinabove, are well-known in the art and include, without limitation, protein A-Sepharose, gel electrophoresis, chromatography, preferably by affinity chromatography, more preferably by affinity chromatography on protein L agarose.

[0394] Another object of the present invention is a composition comprising, consisting essentially of or consisting of at least one fusion protein, at least one nucleic acid encoding said fusion protein, or a at least one expression vector comprising at least one nucleic acid encoding the fusion protein according to the present invention.

[0395] As used herein, “consisting essentially of’, with reference to a composition, means that the at least one fusion protein, nucleic acid or expression vector is the only one therapeutic agent or agent with a biologic activity within said composition.

[0396] Another object of the present invention is a pharmaceutical composition comprising, consisting essentially of or consisting of at least one fusion protein, at least one nucleic acid encoding said fusion protein, or a at least one expression vector comprising at least one nucleic acid encoding the fusion protein according to the present invention, and at least one pharmaceutically acceptable excipient.

[0397] The term “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Said excipient does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a mammal, more preferably a human. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA. [0398] Examples of pharmaceutically acceptable excipients that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0399] In one embodiment, the pharmaceutical compositions according to the present invention comprise vehicles which are pharmaceutically acceptable for a formulation capable of being injected to a subject. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0400] Another object of the present invention is a medicament comprising, consisting essentially of or consisting of at least one fusion protein, at least one nucleic acid encoding said fusion protein, or a at least one expression vector comprising at least one nucleic acid encoding the fusion protein according to the present invention.

[0401] Another object of the present invention is the use of at least one fusion protein, at least one nucleic acid encoding said fusion protein, or a at least one expression vector comprising at least one nucleic acid encoding the fusion protein in the manufacture of a medicament for treating a disease, disorder or symptoms in a subject in need thereof.

[0402] For use in administration to a subject, the composition, pharmaceutical composition or medicament will be formulated for administration to the subject. [0403] In one embodiment, the composition, pharmaceutical composition or medicament according to the present invention is administered (or is to be administered) parenterally, by inhalation spray, rectally, nasally, or via an implanted reservoir.

[0404] In one embodiment, the composition, pharmaceutical composition or medicament is administered (or is to be administered) by injection, including, without limitation, subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection, or infusion techniques.

[0405] Examples of forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms, and the like.

[0406] Sterile injectable forms of the compositions, pharmaceutical compositions or medicaments of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. 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.

[0407] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is to be administered to the subject in need thereof in a therapeutically effective amount.

[0408] It will be however understood that the total daily usage of the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; activity of the fusion protein, nucleic acid, or expression vector employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific fusion protein, nucleic acid, or expression vector employed; the duration of the treatment; drugs used in combination or coincidental with the specific fusion protein, nucleic acid, or expression vector employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered by multiple doses or in a single dose.

[0409] The daily dosage of the fusion proteins, nucleic acids or expression vectors may be varied over a wide range from about 0.01 to about 1000 mg per adult per day. Compositions may contain about 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and about 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A pharmaceutical composition or medicament typically contains from about 0.01 mg to about 500mg of active ingredient.

[0410] In one embodiment, the fusion proteins, nucleic acids or expression vectors of the present invention are to be administered at a dosage level of about 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, or of about 1000 mg per adult per day.

[0411] A therapeutically effective amount of the drug is ordinarily supplied at a dosage level from about 0.0002 mg/kg to about 20 mg/kg of body weight per day. For example, a fusion protein, a nucleic acid or an expression vector present in a composition, pharmaceutical composition or medicament of this invention can be supplied at a concentration ranging from about 1 mg/mL to about 100 mg/mL, such as, for example, at a concentration of about 1 mg/mL, 5 mg/mL, 10 mg/mL, 50 mg/mL or about 100 mg/mL. In one embodiment, the fusion protein, nucleic acid or expression vector is supplied at a concentration of about 10 mg/mL in either 100 mg (10 mL) or 500mg (50mL) single use -vials. It will be appreciated that these dosages are exemplary and that an optimal dosage can be adapted taking into account the affinity and tolerability of the particular fusion protein, nucleic acid or expression vector that must be determined in clinical trials.

[0412] The present invention relates to at least one fusion protein, nucleic acid or expression vector as described herein for use as a medicament, i.e., for treating (or for use in treating) diseases, disorders or symptoms in a subject in need thereof.

[0413] The present invention relates to a composition, pharmaceutical composition, or a medicament as described hereinabove, for use in treating diseases, disorders or symptoms in a subject in need thereof.

[0414] The present invention thus further relates to a method for treating diseases, disorders or symptoms in a subject in need thereof, comprising administering to the subject a fusion protein, a nucleic acid or an expression vector, or a composition, a pharmaceutical composition, or a medicament as described herein.

[0415] Examples of diseases that may be treated in the present invention, include, but are not limited to, cancers, infectious diseases, autoimmune diseases and inflammatory diseases. [0416] In one embodiment, a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition, or a medicament according to the present invention may be used for treating cancer in a subject in need thereof.

[0417] In one embodiment, a fusion protein according to the present invention may be used for treating cancer in a subj ect in need thereof, wherein said fusion protein comprises an antibody or an antigen-binding fragment that induces ADCP, CDC, and/or ADCP activity.

[0418] In one embodiment, a therapeutically effective amount of said fusion protein, nucleic acid or expression vector is administered or is to be administered to the subject.

[0419] 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, without limitation, diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term “cancer” further encompasses both primary and metastatic cancers.

[0420] Examples of cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, endometrial, pancreas or uterus.

[0421] In addition, the cancer may be selected in the following non-limiting list: malignant neoplasm; undifferentiated carcinoma; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma associated with familial polyposis coli; solid carcinoma; malignant carcinoid tumor; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating 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 of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant roblastoma; Sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma - small lymphocytic; malignant diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other 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.

[0422] In another embodiment, a fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or medicament according to the present invention may be used in the treatment of an infectious disease in a subject in need thereof.

[0423] In one embodiment, a therapeutically effective amount of a fusion protein, a nucleic acid or an expression vector according to the present invention is administered or is to be administered to the subject.

[0424] As used herein the term “infectious disease” includes any infection caused by viruses, bacteria, protozoa, molds or fungi.

[0425] In some embodiments, the viral infection comprises infection by one or more viruses selected from the group comprising, but not limited to, Arenaviridae, Astroviridae, Bimaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae or Papillomaviridae viruses. Relevant taxonomic families of RNA viruses include, without limitation, Astroviridae, Bimaviridae, Bromoviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae viruses.

[0426] In some embodiments, the viral infection comprises infection by one or more viruses selected from the group comprising, but not limited to, adenovirus, Alfuy virus, Banzi virus, bovine diarrhea virus, coronavirus, Coxsackie virus, Crimean-Congo virus, Dengue virus, Ebola virus, encephalitis viruses (including Japanese Encephalitis virus, California Encephalitis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Eastern equine encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus), guanarito virus, hantavirus, hepatitis virus, Ilheus virus, immunodeficiency virus, influenza viruses including influenza A and influenza B viruses (including human, avian, and swine) and parainfluenza virus, junin virus, Kokobera virus, Kunjin virus, Kyasanur Forest disease virus, La Crosse virus, Lassa virus, louping-ill virus, lymphocytic choriomeningitis virus, measles virus, machupo virus, Marburg virus, Murray Valley virus, pachindae viruses, Pichinde virus, poliovirus, Powassan virus, Punta Toro virus, respiratory syncytial virus, rhinovirus, Rift Valley Fever virus, Rocio virus, severe acute respiratory syndrome (SARS), small pox virus, Tacaribe virus, West Nile and yellow fever viruses.

[0427] Examples of bacterial infections that may be treated in the present invention include, but are not limited to, infections caused by the following: Staphylococcus; Streptococcus, including S. pyogenes; Enterococci; Bacillus, such as, for example Bacillus anlhracis. and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella such as, for example G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponema; Camplyobacter, Pseudomonas such as, for example, P. aeruginosa; Legionella; Neisseria such as, for example N. gonorrhoeae and N. meningitides; Flavobacterium such as, for example F. meningosepticum and F. odoraturn; Brucella; Bordetella such as, for example B. pertussis and B. bronchiseptica; Escherichia such as, for example E. coll. Klebsiella; Enterobacter, Serratia such as, for example S. marcescens and S. liquefaciens; Edwardsiella; Proteus such as, for example P. mirabilis and P. vulgaris; Streptobacillus; Rickettsiaceae such as, for example R. fickellsfi. Chlamydia such as, for example C. psittaci and C. trachomatis; Mycobacterium such as, for example M. tuberculosis, M. intracellulare , M. folluiturn, M. laprae, M. avium, M. bovis, M. africanum, M. kansasii, and M. lepraernuriunr, and Nocardia.

[0428] Examples of protozoa infections that may be treated in the present invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosoma.

[0429] A complete list of infectious diseases can be found on the website of the National Center for Infectious Disease (NCID) at the Center for Disease Control (CDC) (World Wide Web (www) at cdc.gov/ncidod/diseases/), which list is incorporated herein by reference. All of said diseases are candidates for treatment using the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the invention.

[0430] In one embodiment, a fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or medicament according to the present invention may be used in the treatment of an autoimmune disease in a subject in need thereof.

[0431] In one embodiment, a fusion protein according to the present invention may be used for treating an autoimmune disease in a subject in need thereof, wherein said fusion protein comprises an antibody or an antigen-binding fragment thereof that does not induce ADCC, ADCP and/or CDC.

[0432] In one embodiment, a therapeutically effective amount of a fusion protein, a nucleic acid or an expression vector according to the present invention is administered or is to be administered to the subject.

[0433] As used herein, the term “autoimmune diseases” refers to diseases with defects in the immune system causing an immune response to self-tissue.

[0434] Examples of autoimmune diseases include, but are not limited to, lupus (e.g., lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, Wegener's disease; primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison’s disease, diabetes (e.g. insulin dependent diabetes mellitus, type I diabetes mellitus, type II diabetes mellitus), good pasture’s syndrome, myasthenia gravis, pemphigus, intestinal inflammatory conditions such as Crohn’s disease and ulcerative colitis; sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulcerative colitis, Sjogren’s syndrome, arthritis conditions such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and juvenile idiopathic arthritis; polymyositis, scleroderma, psoriasis, primary sclerosing cholangitis; asthma, transplant rejection (host versus graft disease); graft versus host disease and mixed connective tissue disease.

[0435] In one embodiment, a fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or medicament according to the present invention may be used in the treatment of an inflammatory disease in a subject in need thereof.

[0436] In one embodiment, a fusion protein according to the present invention may be used for treating an inflammatory disease in a subject in need thereof, wherein said fusion protein comprises an antibody or an antigen-binding fragment thereof that does not induce ADCC, ADCP and/or CDC.

[0437] In one embodiment, a therapeutically effective amount of a fusion protein, a nucleic acid or an expression vector according to the present invention is administered or is to be administered to the subject.

[0438] As used herein, the term “inflammatory diseases” includes a vast array of disorders and conditions that are characterized by inflammation.

[0439] Examples of inflammatory diseases include, but are not limited to, arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, arthritis uratica, gout, chronic polyarthritis, periarthritis humeroscapularis, cervical arthritis, lumbosacral arthritis, enteropathic arthritis and ankylosing spondylitis, asthma, dermatitis, psoriasis, scleroderma, polymyositis, dermatomyositis, juvenila dermatomyositis, primary biliary cirrhosis, fibrosis, cystic fibrosis, pulmonary fibrosis, cirrhosis, endomyocardial fibrosis, dediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, nephrogenic fibrosis, Keloids, scleroderma, arthrofibrosis, post transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, lupus nephritis, pemphigus, Pemphigus vulgaris, Pemphigus herpetiformis, Pemphigus vegetans, IgA pemphigus, Pemphigus erythematosus, bullous pemphigoid, Pemphigoid gestationis, Mucous membrane dermatosis, Pemphigoid nodularis, Linear IgA bullous dermatosis, Bullous lichen planus, Epidermolysis bullosa acquisita, autoimmune diabetes, diabetic retinopathy, diabetic nephropathy, diabetic vasculopathy, ocular inflammation, uveitis, rhinitis, ischemiareperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), glomerulonephritis, Graves disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, angina, small artery disease, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiple sclerosis, systemic sclerosis, antiphospholipid syndrome, Sjoegren’s syndrome, autoimmune hemolytic anemia, colitis, Crohn’s Disease, ulcerative colitis, Inflammatory Bowel Disease (IBD), embolism, pulmonary embolism, arterial embolism, venous embolism, allergic inflammation, cardiovascular disease, graft- related diseases, graft versus host disease (GVHD), disorders associated with graft transplantation rejection, chronic rejection, and tissue or cell allografts or xenografts, autoimmune diseases, degeneration after trauma, stroke, transplant rejection, allergic conditions and hypersensitivity, e.g., allergic rhinitis, allergic eczema and the like, skin diseases, dermal inflammatory disorders, or any combination thereof.

[0440] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used alone.

[0441] In another embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with at least one further therapeutic agent.

[0442] In one embodiment, the administration of the at least one further therapeutic agent and of the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is simultaneous, separate or sequential.

[0443] In one embodiment, for simultaneous administration, the at least one further therapeutic agent and the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention are administered as one composition or as separate compositions, as appropriate.

[0444] Examples of additional therapeutic agents include, but are not limited to, chemotherapeutic agents, targeted cancer therapy, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti- angiogenic agents, cell cycle control/apoptosis regulating agents, hormonal regulating agents, and other immunosuppressive and/or anti-inflammatory drugs selected from corticoids, such as, for example, glucocorticoids.

[0445] In one embodiment, the at least one further therapeutic agent is a therapeutic agent useful for treating the specific disease, disorder or condition to be treated in the present invention. For example, for treating cancer, the at least one further therapeutic agent may be selected from the group comprising, but not limited to, chemotherapeutic agents, targeted cancer therapy, radiotherapy, immunotherapeutic agents or anti-cancer immunogens, anti-cancer antibodies, cytotoxic agents, anti-angiogenic agents, cell cycle control/apoptosis regulating agents, hormonal regulating agents, and other immunosuppressive and/or anti-inflammatory drugs selected from corticoids, such as, for example, glucocorticoids.

[0446] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a chemotherapeutic agent.

[0447] The term “chemotherapeutic agent” refers to chemical compounds that are effective in inhibiting tumor growth.

[0448] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin 11 and calicheamicin 211; dynemicin, including dynemicin A; an esperamicin; neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0449] Also included in the definition of chemotherapeutic agents are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0450] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a targeted cancer therapy.

[0451] As used herein, the term “targeted cancer therapies” are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules (“molecular targets”) that are involved in the growth, progression, and spread of cancer. Targeted cancer therapies are sometimes called “molecularly targeted drugs”, “molecularly targeted therapies”, “precision medicines”. In some embodiments, the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor. The term “tyrosine kinase inhibitor” refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, ABT- 869, AEE-788, AEW-541, Axitinib, AZM-475271, BEZ235, BMS-599626 (AC-480), Bosutinib, Brivanib (BMS-582664), canertinib (CI 1033), Cediranib, CEP-11981, CP- 547632, CP-724714, dasatinib (BMS-354825), Dovitinib, Enzastaurin, erlotinib (Tarceva; OSI-1774), gefitinib (Iressa), imatinib (Gleevec; STI571), KRN-633, KRN- 951, lapatinib (GW572016; GW2016), leflunomide (SU101), Lestaurtinib, L-21649, Motasenib, Midostaurin, MKC-I (Ro-317453; R-440), MK-2206 (8-[4- aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4-f][l,6]naphthyridin-3(2H)-one hydrochloride), MLN-8054, Neratinib, Nilotinib, OSI-930, Pazopanib, PD-0325901, PD- 0332991, PP2, saracatinib, semaxinib (SU5416), Seliciclib, SNS-032, sorafenib (BAY 43-9006), sunitinib (Sutent; SU11248), SU-14813, SU-6668 (TSU-68), TAK-165, Tandutinib, Telatinib, vatalanib (PTK787/ZK222584), vandetanib (Zactima; ZD6474), derivatives thereof, analogs thereof, and combinations thereof.

[0452] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with radiotherapy.

[0453] The term “radiotherapy” may comprise radiation or associated administration of radiopharmaceuticals to a patient. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-i l l.

[0454] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an immunotherapeutic agent or immunotherapy.

[0455] The terms “immunotherapeutic agent” or “immunotherapy” as used herein, refer to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body’s immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system’s responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy, biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents or immunotherapies known in the art include, but are not limited to: cytokines, checkpoint inhibitors, checkpoint agonists also referred to as T cell agonists, antibodies including monoclonal antibodies, antibody domains, antibody fragments, bispecific antibodies, preventive and therapeutic vaccines, oncolytic viruses, adoptive transfer of immune cells (T cells, NK, cells, dendritic cells, B cells. . .).

[0456] One of the central premises underlying cancer immunotherapy is the presence of antigens which are selectively or abundantly expressed or mutated in cancer cells, thus enabling the specific recognition and subsequent destruction of the cancer cells. Such antigens are commonly referred to as tumor-specific antigens. Another of the central premises underlying cancer immunotherapy is the presence of lymphocytes in the tumors, ie., tumor infiltrating lymphocytes (TILs), and notably of effector TILs which can target and kill the tumor cells through the recognition of the above-mentioned tumor-specific antigens.

[0457] Immunotherapeutic agents or therapies can be passive. A passive immunotherapeutic agent is one that produces an immediate action due to the administration of immune-cell factors, like monoclonal antibodies. The results of a passive immunotherapy are tied temporally to administration of the agent, therefore continued dosing may be required for a prolonged response. In another embodiment, the immunotherapeutic agent or therapies are active. An active immunotherapeutic agent is one that produces a lasting, durable response by way of inducing immunological memory. This most closely resembles a normal immune response. However, just as immune system function varies in a healthy population, the level of response to an active immunotherapy agent depends on individual factors.

[0458] Active immunotherapeutic agents include both non-specific active agents (i.e., agents that boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells), and specific active agents, (i.e., agents inducing the generation of cell-mediated and antibody immune responses focused on specific antigens expressed by the cancer cells). Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g., cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.

[0459] In one embodiment, the fusion protein, nucleic acid or expression vector according to the present invention is used in combination with a cytokine therapy.

[0460] As used herein, a “cytokine therapy” is defined as the administration of at least one cytokine to the subject.

[0461] A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins (except interleukins that are from the common cytokine receptor gamma-chain family) and colony-stimulating factors.

[0462] Common cytokine receptor gamma-chain family consists of six members, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Therefore, in one embodiment, suitable interleukins that may be used in the present invention include, without limitation cytokines of the IL- 1, IL-6, IL- 10, IL- 12 and IL- 17 families, Th2-like cytokines, cytokines of the transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), growth differentiation factor (GDF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) families, neurotrophins, angiopoietins, cytokines of the tumor necrosis factor (TNF) family, cytokines of the interferon (IFN) family, cytokines of the colony-stimulating factors (CSF) family and other cytokines, such as, for example, insulin-like growth factor (IGF), prolactin (PRL) and sonic hedgehog (SHH).

[0463] Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN-gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognize and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).

[0464] Colony-stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. I l l

Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Amesp (erytropoietin).

[0465] In one embodiment, the fusion protein, nucleic acid or vector according to the present invention is used in combination with a cytokine mimetic.

[0466] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a checkpoint inhibitor therapy.

[0467] As used herein, a “checkpoint inhibitor therapy” is defined as the administration of at least one checkpoint inhibitor to the subject.

[0468] As a cancer treatment, checkpoint inhibitor therapy aims at preventing the activation of inhibitory receptors expressed on T cells by ligands expressed by the tumor cells. Checkpoint inhibitor therapy thus aims at preventing the inhibition of T cells present in the tumor, z.e., tumor infiltrating T cells, and thus at enhancing the subject immune response towards the tumor cells.

[0469] Examples of checkpoint inhibitors include, without being limited to, inhibitors of the cell surface receptor PD-1 (programmed cell death protein 1), also known as CD279 (cluster differentiation 279); inhibitors of the ligand PD-L1 (programmed death-ligand 1), also known as CD274 (cluster of differentiation 274) or B7-H1 (B7 homolog 1); inhibitors of the cell surface receptor CTLA4 or CTLA-4 (cytotoxic T-lymphocyte- associated protein 4), also known as CD 152 (cluster of differentiation 152); inhibitors of LAG-3 (lymphocyte-activation gene 3), also known as CD223 (cluster differentiation 223); inhibitors of TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), also known as HAVCR2 (hepatitis A virus cellular receptor 2) or CD366 (cluster differentiation 366); inhibitors of TIGIT (T cell immunoreceptor with Ig and ITIM domains), also known as VSIG9 (V-Set And Immunoglobulin Domain-Containing Protein 9) or VSTM3 (V-Set And Transmembrane Domain-Containing Protein 3); inhibitors of BTLA (B and T lymphocyte attenuator), also known as CD272 (cluster differentiation 272); inhibitors of CEACAM-1 (carcinoembryonic antigen-related cell adhesion molecule 1) also known as CD66a (cluster differentiation 66a).

[0470] In one embodiment, the at least one checkpoint inhibitor is selected from the group comprising or consisting of inhibitors of PD-1, inhibitors of PD-L1, inhibitors of CTLA-4 and any mixtures thereof.

[0471] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a checkpoint agonist therapy.

[0472] As used herein, a “checkpoint agonist therapy” is defined as the administration of at least one checkpoint agonist to the subject.

[0473] As a cancer treatment, checkpoint agonist therapy aims at activating stimulatory receptors expressed on immune cells present in a tumor. In particular, T-cell agonist therapy aims at enhancing the activation of T cells present in a tumor, /.< ., tumor infiltrating T cells, and thus at enhancing the subject immune response towards the tumor cells. Currently, a number of potential targets for checkpoint agonist therapy have been identified.

[0474] Examples of checkpoint agonists include, without being limited to, agonists of CD137 (cluster differentiation 137) also known as 4-1BB or TNFRS9 (tumor necrosis factor receptor superfamily, member 9); agonists of 0X40 receptor also known as CD 134 (cluster differentiation 134) or TNFRSF4 (tumor necrosis factor receptor superfamily, member 4); agonists of GITR (glucocorticoid-induced TNF receptor family-related protein); agonists of ICOS (inducible co-stimulator); agonists of CD27-CD70 (cluster differentiation 27-cluster differentiation 70); and agonists of CD40 (cluster differentiation 40).

[0475] In one embodiment, the at least one checkpoint agonist is selected from the group comprising or consisting of agonists of CD 137, agonists of 0X40, agonists of GITR, agonists of ICOS, agonists of CD27-CD70, agonists of CD40 and any mixtures thereof. [0476] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an antibody that is specific for an immune receptor or a costimulatory molecule.

[0477] Examples of antibodies that are specific for an immune receptor include but are not limited to anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-B7H6 antibodies, anti-4- IBB antibodies, anti-TIGIT antibodies, anti-ICOS antibodies, anti-GITR antibodies, anti-CD27-CD70 antibodies, anti-CD40 antibodies, anti-BTLA antibodies, anti-HVEM antibodies, anti-CD160 antibodies, anti-CCR8 antibodies, anti-CEACAM-1 and anti-OX40 antibodies.

[0478] In some embodiments, the antibody is specific for CD137. As used herein the term “CD 137” has its general meaning in the art and may also be referred to as Ly63, ILA or 4-1BB. CD137 is a member of the tumor necrosis factor (TNF) receptor family. Members of this receptor family and their structurally related ligands are important regulators of a wide variety of physiologic processes and play an important role in the regulation of immune responses. CD137 is expressed by activated NK cells, T and B lymphocytes and monocytes/macrophages. The gene encodes a 255-amino acid protein with 3 cysteine-rich motifs in the extracellular domain (characteristic of this receptor family), a transmembrane region, and a short N- terminal cytoplasmic portion containing potential phosphorylation sites. Expression in primary cells is strictly activation dependent. The ligand for the receptor is TNFSF9. Human CD 137 is reported to bind only to its ligand. Agonists include the native ligand (TNFSF9), aptamers (see McNamara et al. (2008) J. Clin. Invest. 1 18: 376-386), and antibodies.

[0479] In another embodiment, the antibody or antigen-binding fragment thereof is bispecific, and is further capable of binding to an immune receptor or to a costimulatory molecule. [0480] Examples of immune receptors include, but are not limited to, CTLA4, PD-1, PD-L1, TIM-3, LAG-3, B7H3, B7H4, B7H6, 4-1BB, TIGIT, ICOS, GITR, CD27-CD70, CD40, BTLA, HVEM, CD 160, CCR8, CEACAM-1 and 0X40.

[0481] Examples of costimulatory molecules include, but are not limited to, B7H3, B7H4, B7H6, 4- IBB and 0X40, GITR.

[0482] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an antibody that induces, via ADCC, the death of a cell expressing an antigen to which the antibody binds. In one embodiment, the antibody (e.g. of IgGl or IgG3 isotype, in particular of human IgGl or IgG3 isotype) induces ADCC toward a cell to which the antibody binds. NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such an antibody. In one embodiment, the antibody is specific for a cell surface antigen, e.g., membrane antigen. In some embodiments, the antibody is specific for a tumor antigen (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, aVp3, etc., particularly lymphoma antigens (e.g., CD20).

[0483] Accordingly, the present invention also provides methods to enhance the antitumor effect of monoclonal antibodies directed against tumor antigen(s).

[0484] In one embodiment, ADCC function is specifically augmented, which in turn enhances target cell killing, by sequential administration of an antibody directed against one or more tumor antigens, and a fusion protein according to the present invention.

[0485] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a natural ligand of an NK cell activating receptor or an antibody that binds and activates an NK cell activating receptor. [0486] In one embodiment, the at least one further therapeutic agent is an agent that increases the presence of a natural ligand of an NK cell activating receptor on the surface of a target cell (e.g., infected cells, or tumor cells).

[0487] As used herein, the term “activating NK receptor” refers to any molecule on the surface of NK cells that, when stimulated, causes a measurable increase in any property or activity known in the art as associated with NK activity, such as cytokine (for example IFN-y and TNF-a) production, increases in intracellular free calcium levels, the ability to target cells in a redirected killing assay, or the ability to stimulate NK cell proliferation.

[0488] Examples of “activating NK receptors” include but are not limited to activating forms of KIR proteins (for example KIR2DS proteins), CD160-TM, NKG2D, IL-2R, IL- 12R, IL-15R, IL-18R and IL-21R.

[0489] Examples of ligands that act as agonists at activating receptors include, e.g. IL- 2, IL-15, IL-21 polypeptides.

[0490] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a therapeutic vaccine or treatment vaccine.

[0491] As used herein, a therapeutic vaccine is defined as the administration of at least one tumor-specific antigen (e.g., synthetic long peptides or SLP), or of the nucleic acid encoding said tumor-specific antigen; the administration of recombinant viral vectors selectively entering and/or replicating in tumor cells; the administration of tumor cells; and/or the administration of immune cells (e.g., dendritic cells) engineered to present tumor-specific antigens and trigger an immune response against these antigens.

[0492] As a cancer treatment, therapeutic vaccines aim at enhancing the subj ect immune response towards the tumor cells.

[0493] Examples of therapeutic vaccines aiming at enhancing the subject immune response towards tumor cells include, without being limited to, viral-vector based therapeutic vaccines such as adenoviruses (e.g., oncolytic adenoviruses), vaccinia viruses (e.g., modified vaccinia Ankara (MV A)), alpha viruses (e.g., Semliki Forrest Virus (SFV)), measles virus, Herpes simplex virus (HSV), and coxsackievirus; synthetic long peptide (SLP) vaccines; RNA-based vaccines, and dendritic cell vaccines.

[0494] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an oncolytic virus therapy.

[0495] As used herein, an “oncolytic virus therapy” is defined as the administration of at least one oncolytic virus to the subject.

[0496] Oncolytic viruses are defined as viruses that preferentially infect and kill cancer cells over normal, non-cancer, cells. As a cancer treatment, oncolytic virus therapy aims at killing cancer cells and/or triggering or enhancing an immune response towards the cancer cells.

[0497] Examples of oncolytic viruses include, without being limited to, modified herpes simplex type-1 viruses such as talimogene laherparepvec (or T-VEC) or HSV-1716; modified adenoviruses such as Ad5-DNX-2401; modified measles viruses such as MV- NIS; modified vaccinia viruses (W) such as vaccinia virus TG6002; and modified polioviruses such as PVS-RIPO.

[0498] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an adoptive transfer of cells, also referred to as adoptive cell therapy (both also referred to as ACT), such as, for example, an adoptive transfer of T cells or NK cells, also referred to as adoptive T cell therapy or adoptive NK cell therapy, respectively.

[0499] As used herein, an “adoptive transfer of cells” or “adoptive cell therapy” is defined as the transfer, for example as an infusion or re-infusion, of immune cells to a subject. As a cancer treatment, the adoptive transfer of immune cells to a subject aims at enhancing the subject immune response towards the cancer cells.

[0500] Examples of immune cells that may be used for a cell therapy include without limitation cytotoxic cells (e.g., natural killer (NK) cells, CD8⁺ T cells, and natural killer (NK) cells T cells), effector T cells (e.g., CD4⁺ T cells and CD8⁺ T cells), alpha beta (aP) T cells, and gamma delta (y6) T cells, antibody-expressing B cells or other antibodyproducing or -presenting cells and dendritic cells.

[0501] In one embodiment, the transferred immune cells as described hereinabove are antigen-specific cells. In one embodiment, the transferred immune cells as described hereinabove are antigen-specific immune cells, wherein said antigen is specifically and/or abundantly expressed by cancer cells. In one embodiment, the transferred immune cells as described hereinabove are tumor-specific immune cells, in other words the transferred immune cells as described hereinabove specifically recognize cancer cells or tumor cells through an antigen specifically and/or abundantly expressed by said cancer cells or tumor cells. In one embodiment, the transferred immune cells as described hereinabove are tumor-specific effector T cells. In one embodiment, the transferred immune cells as described hereinabove are tumor-specific CD8⁺ effector T cells, in particular tumor-specific cytotoxic CD8⁺ T cells. In one embodiment, the transferred cells are tumor infiltrating cells (TIL). In one embodiment, the transferred immune cells as described hereinabove are tumor-specific cytotoxic cells. In one embodiment, the transferred immune cells as described hereinabove are tumor-specific NK cells.

[0502] Examples of tumor-specific antigens, z.e.., antigens that are specifically and/or abundantly expressed by cancer cells include, without being limited to, neoantigens (also referred to as new antigens or mutated antigens), 9D7, ART4, P-catenin, BING-4, Bcr- abl, BRCA1/2, calcium-activated chloride channel 2, CDK4, CEA (carcinoembryonic antigen), CML66, Cyclin Bl, CypB, EBV (Epstein-Barr virus) associated antigens (such as LMP-1, LMP-2, EBNA1 and BARF1), Ep-CAM, EphA3, fibronectin, Gpl00/pmell7, Her2/neu, HPV (human papillomavirus) E6, HPV E7, hTERT, IDH1, IDH2, immature laminin receptor, MC1R, Melan-A/MART-1, MART-2, mesothelin, MUC1, MUC2, MUM-1, MUM-2, MUM-3, NY-ESO-l/LAGE-2, p53, PRAME, prostate-specific antigen (PSA), PSMA (prostate-specific membrane antigen), Ras, SAP-1, SART-I, S ART-2, SART-3, SSX-2, survivin, TAG-72, telomerase, TGF- PRII, TRP-1/-2, tyrosinase, WT1, antigens of the BAGE family, antigens of the CAGE family, antigens of the GAGE family, antigens of the MAGE family, antigens of the SAGE family, and antigens of the XAGE family.

[0503] As used herein, neoantigens (also referred to as new antigens or mutated antigens) correspond to antigens derived from proteins that are affected by somatic mutations or gene rearrangements acquired by the tumors. Neoantigens may be specific to each individual subject and thus provide targets for developing personalized immunotherapies. Examples of neoantigens include for example, without being limited to, the R24C mutant of CDK4, the R24L mutant of CDK4, KRAS mutated at codon 12, mutated p53, the V599E mutant of BRAF and the R132H mutant of IDH1.

[0504] In one embodiment, the transferred immune cells as described hereinabove are specific for a tumor antigen selected from the group comprising or consisting of the class of CTAs (cancer/testis antigens, also known as MAGE-type antigens), the class of neoantigens and the class of viral antigens.

[0505] As used herein, the class of CTAs corresponds to antigens encoded by genes that are expressed in tumor cells but not in normal tissues except in male germline cells. Examples of CTAs include, without being limited to, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C2, NY-ESO-1, PRAME and SSX-2.

[0506] As used herein, the class of viral antigens corresponds to antigens derived from viral oncogenic proteins. Examples of viral antigens include, without being limited to, HPV (human papillomavirus) associated antigens such as E6 and E7, and EBV (Epstein-Barr virus) associated antigens such as LMP-1, LMP-2, EBNA1 and BARF1.

[0507] In one embodiment, the transferred immune cells as described hereinabove are autologous immune cells, in particular autologous T cells. In another embodiment, the transferred immune cells as described hereinabove are allogenic (or allogenous) immune cells, in particular allogenic NK cells. [0508] For example, autologous T cells can be generated ex vivo either by expansion of antigen-specific T cells isolated from the subject or by redirection of T cells of the subject through genetic engineering.

[0509] In one embodiment, the immune cells to be infused are modified ex vivo before being infused to the subject.

[0510] Methods to isolate T cells from a subject, in particular antigen-specific T cells, e.g., tumor-specific T cells, are well-known in the art (see for example Rosenberg & Restifo, 2015, Science 348, 62-68; Prickett et al., 2016, Cancer Immunol Res 4, 669-678; or Hinrichs & Rosenberg, 2014, Immunol Rev 257, 56-71). Methods to expand T cells ex vivo are well-known in the art (see for example Rosenberg & Restifo, 2015, Science 348, 62-68; Prickett et al., 2016, Cancer Immunol Res 4, 669-678; or Hinrichs & Rosenberg, 2014, Immunol Rev 257, 56-71). Protocols for infusion of T cells in a subject, including pre-infusion conditioning regimens, are well-known in the art (see for example Rosenberg & Restifo, 2015, Science 348, 62-68; Prickett et al., 2016, Cancer Immunol Res 4, 669-678; or Hinrichs & Rosenberg, 2014, Immunol Rev 257, 56-71).

[0511] In one embodiment, the fusion protein, nucleic acid, expression vector, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with a CAR immune cell therapy, in particular a CAR T cell therapy or a CAR NK cell therapy.

[0512] As used herein, CAR immune cell therapy is an adoptive cell therapy wherein the transferred cells are immune cells as described hereinabove, such as T cells or NK cells, genetically engineered to express a chimeric antigen receptor (CAR). As a cancer treatment, the adoptive transfer of CAR immune cells to a subject aims at enhancing the subject immune response towards the cancer cells.

[0513] CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule or in several molecules. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moi eties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are usually derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and antitumor activity in vivo. Thus, signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells.

[0514] Thus, in one embodiment, the transferred T cells as described hereinabove are CAR T cells. The expression of a CAR allows the T cells to be redirected against a selected antigen, such as an antigen expressed at the surface of cancer cells. In one embodiment, the transferred CAR T cells recognize a tumor-specific antigen.

[0515] In another embodiment, the transferred NK cells as described hereinabove are CAR NK cells. The expression of a CAR allows the NK cells to be redirected against a selected antigen, such as an antigen expressed at the surface of cancer cells. In one embodiment, the transferred CAR NK cells recognize a tumor-specific antigen.

[0516] Examples of tumor-specific antigens are mentioned hereinabove.

[0517] In one embodiment, the transferred CAR T cells or CAR NK cells recognize a tumor-specific antigen selected from the group comprising or consisting of EGFR and in particular EGFRvIII, mesothelin, PSMA, PSA, CD47, CD70, CD133, CD171, CEA, FAP, GD2, HER2, IL-13Ra, avp6 integrin, ROR1, MUC1, GPC3, EphA2, CD19, CD21, and CD20.

[0518] In one embodiment, the CAR immune cells as described hereinabove are autologous CAR immune cells, in particular autologous CAR T cells. In another embodiment, the CAR immune cells as described hereinabove are allogenic (or allogenous) CAR immune cells, in particular allogenic CAR NK cells. [0519] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an antibiotic. Examples of antibiotics include, but are not limited to, penicillins (e.g., penicillin, amoxicillin), tetracyclines (e.g., doxy cy client, tetracycline, minocycline), cephalosporins (e.g., cefuroxime, ceftriaxone, cefdinir), quinolones (e.g., ciprofloxacin, levofloxacin, moxifloxacin), lincomycins (e.g., clindamycin, lincomycin), macrolides (e.g., azithromycin, clarithromycin, erythromycin), sulfonamides (e.g., sulfamethoxazole-trimethoprim, sulfasalazine, sulfisoxazole), glycopeptides (e.g., dalbavancin, oritavancin, telavancin, vancomycin), aminoglycosides (e.g., gentamicin, tobramycin, amikacin) and carbapenems (e.g., imipenem, meropenem, doripenem, ertapenem).

[0520] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an antiviral drug. Examples of antiviral drugs include, but are not limited to, abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliever famciclovir, fomivirsen, fosamprenavir, foscamet, fosfonet, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogues, norvir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, raltegravir, reverse transcriptase inhibitor, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0521] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an antifungal agent. Examples of antifungal agents include, but are not limited to, polyene antifungals (e.g., amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, rimocidin), imidazole antifungals (e.g., bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole), triazole antifungals (e.g., albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propi conazole, ravuconazole, terconazole, voriconazole), thiazole antifungals (e.g., abafungin), allylamines, echinocandins (e.g., anidulafungin, caspofungin, micafungin).

[0522] In one embodiment, the fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition or medicament according to the present invention is used in combination with an anti-parasitic agent. Examples of anti-parasitic agents include, but are not limited to, broad-spectrum anti-parasitic agents (e.g., nitazoxanide), antiprotozoal s (e.g., melarsoprol, eflomithine, metronidazole, tinidazole, miltefosine), antihelminthic (including, without limitation, antinematodes (ancylostoma caninum, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin), anticestodes (e.g., niclosamide, praziquantel, albendazole), antitrematodes (e.g., praziquantel)), antiamoebics (e.g., rifampin, amphotericin B).

[0523] Another object of the present invention relates to the use of a fusion protein, nucleic acid or expression vector as described hereinabove (preferably in a composition, pharmaceutical composition or medicament) with another therapeutic agent as described hereinabove, in the treatment of diseases in a subject in need thereof, wherein said fusion protein, nucleic acid or expression vector is used as an adjuvant for the therapeutic agent.

[0524] The present invention thus relates to a fusion protein nucleic acid or expression vector as described hereinabove (preferably in a composition, pharmaceutical composition or medicament), for use as an adjuvant in a cancer therapy. The present invention thus relates to a fusion protein, nucleic acid or expression vector as described hereinabove (preferably in a composition, pharmaceutical composition or medicament), for use as an adjuvant in a therapy for an infectious disease. [0525] As used herein, the term “adjuvant” refers to a compound or a combination of compounds that potentiates at therapy, such as, for example, a cancer therapy. Adjuvants may increase the effective immune response against low or non-immunogenic tumor cells. In one embodiment, the adjuvant is used with a well-known cancer therapeutic agent in the treatment of cancer and thus potentiates the immune response towards cancer cells. For example, an adjuvant may potentiate an immune response during a cancer therapy, decrease T cell exhaustion (without decreasing T cells activation), increase the survival of T cells, enhance NK cells cytotoxicity, decrease the tumor growth and/or the tumor size, and/or increase in survival, treats or prevents cancer metastasis. In one embodiment, potentiation of a cancer therapy in the presence of an adjuvant, is defined by comparison with a cancer therapy administered alone.

[0526] In one embodiment, the present invention relates to the use of a fusion protein nucleic acid or expression vector as described hereinabove (preferably in a composition, pharmaceutical composition or medicament as described hereinabove), for potentiating an immune response induced by a cancer therapy in a patient in need thereof.

[0527] Thus, the present invention further relates to a method comprising the step of administering to a subject a cancer therapy, wherein the subject has received or is receiving a therapeutically effective amount of a fusion protein, nucleic acid, expression vector, composition, pharmaceutical composition, or medicament as disclosed herein.

[0528] In one embodiment, the fusion protein, nucleic acid or expression vector according to the present invention (preferably in a composition, pharmaceutical composition or medicament as described hereinabove) may be used as immunotherapeutic agent, particularly to treat a wide variety of cancers (e.g., cancers associated with immunosuppression and/or immune exhaustion).

[0529] In one embodiment, the fusion protein, nucleic acid or expression vector according to the present invention (preferably in a composition, pharmaceutical composition or medicament as described hereinabove) may potentiate an immune response induced by a cancer therapy in a patient by administering said fusion protein, nucleic acid or expression vector to a subject in an amount effective to potentiate an immune response induced by the cancer therapy in the patient.

[0530] In another embodiment, a fusion protein, nucleic acid or expression vector as described hereinabove (preferably in a composition, pharmaceutical composition or medicament as described hereinabove) can increase or improve the immune response of a subject.

[0531] As used herein, an “immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen- presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. In some embodiments of any of the aspects, the response is specific for a particular antigen (an “antigen-specific response”), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments of any of the aspects, an immune response is a T cell response, such as a CD4⁺ response or a CD8⁺ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.

[0532] As with other known immunotherapeutic agents, the ability of a fusion protein, nucleic acid or expression vector as described hereinabove, to potentiate an immune response in a patient may have broader therapeutic implications outside the cancer field. For example, it has been proposed that immune potentiating agents may be useful in treating a wide variety of infectious diseases, particularly pathogenic agents which promote immunosuppression and/or immune exhaustion. Also, such immune potentiating agents may be useful in boosting the immunization efficacy of vaccines (e.g., infectious disease and cancer vaccines).

[0533] Another object of the present invention relates to the use of a fusion protein as described hereinabove, or of a nucleic acid or an expression vector according to the present invention (preferably in a composition, pharmaceutical composition or medicament as described hereinabove) to deplete CD25 expressing Treg cells in a subject in need thereof, wherein a therapeutically effective amount of the fusion protein, the nucleic acid, or of the expression vector of the present invention is to be administered to the subject.

[0534] The present invention thus further relates to a method for depleting CD25 expressing Treg cells in a subject in need thereof, comprising administering to the subject a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition, or a medicament as described herein.

[0535] In one embodiment, the fusion protein, the nucleic acid or the expression vector as described hereinabove (preferably in a composition, pharmaceutical composition or medicament as described hereinabove), is for use to deplete CD25 expressing Treg cells.

[0536] In one embodiment, the CD25 expressing Treg cells are tumor infiltrating Tregs.

[0537] In one embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein as described hereinabove for use to deplete CD25 expressing Treg cells is an IgG, preferably an IgGl .

[0538] In one embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein as described hereinabove for use to deplete CD25 expressing Treg cells binds to at least one activating Fey Receptor, preferably selected from FcyRI, FcyRIIa, FcyRIII with a high affinity.

[0539] In one embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein as described hereinabove for use to deplete CD25 expressing Treg cells elicits an enhanced ADCC, ADCP and/or CDC response, preferably an increased ADCC and/or ADCP response, more preferably an increased ADCC response.

[0540] In one embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein as described hereinabove for use to deplete CD25 expressing Treg cells does not inhibit the IL-2 signaling via CD25. Thus, in one embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein as described hereinabove for use to deplete CD25 expressing Treg cells does not inhibit the proliferation and/or activation of CD4⁺ and CD8⁺ T cells (or effector T cells). In another embodiment, the antibody or antigen-binding fragment thereof comprised in a fusion protein as described hereinabove for use to deplete CD25 expressing Treg cells does not inhibit the phosphorylation of STAT5a in CD4⁺ and CD8⁺ T cells (or effector T cells).

[0541] The present invention further relates to a method of inducing specific lysis of CD25 positive cells without inhibiting IL-2 signaling in T-cells, the method comprising the step of administering to a subject a therapeutically effective amount of a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition, or a medicament as disclosed herein.

[0542] The present invention further relates to a method of inducing specific lysis of CD25 positive cells by ADCC and/or ADCP without inhibiting IL-2 signaling in T-cells, the method comprising the step of administering to a subject a therapeutically effective amount of an isolated protein a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition, or a medicament as disclosed herein.

[0543] In some embodiments, the subject is receiving or has received an immunotherapy.

[0544] The present invention further relates to a method comprising the step of administering to a subject an immunotherapy, wherein the subject has received or is receiving a therapeutically effective amount of a fusion protein, a nucleic acid, an expression vector, a composition, a pharmaceutical composition, or a medicament as disclosed herein.

[0545] In some embodiments, the therapeutically effective amount is an amount effective to induce specific lysis of CD25 positive cells without inhibiting IL-2 signaling in T-cells.

[0546] In some embodiments, the therapeutically effective amount is an amount effective to induce specific lysis of CD25 positive cells by ADCC and/or ADCP without inhibiting IL-2 signaling in T-cells.

[0547] The present invention further relates to the use of a fusion protein, a nucleic acid, a, expression vector as disclosed herein in the manufacture of a medicament for treating diseases, disorders or symptoms as disclosed hereinabove (e.g. cancer or infectious diseases) in a subject in need thereof.

[0548] As compared to the fusion proteins or antibodies of the prior art, the fusion proteins of the present invention, and in particular, the fusion proteins comprising the anti-CD25 antibodies or antigen-binding fragments thereof as disclosed herein, may present at least one of the following advantages: in some embodiments, the fusion proteins of the present invention present an increased affinity for CD25, as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention present an increased avidity for CD25, as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention induce an increased IL-2-dependent activation of T cells in culture, and preferably an increased proliferation of T cells in culture, as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention induce a lower inhibition of IL-2-induced T cell proliferation in culture as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention present an increased ADCC activity on CD25⁺ expressing cells, preferably CD25⁺ expressing T cells and more preferably CD25⁺ expressing Treg cells as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention present an increased ADCP activity on CD25⁺ expressing cells, preferably CD25⁺ expressing T cells and more preferably CD25⁺ expressing Treg cells as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention induce depletion (preferably in vivo depletion) of CD25⁺ expressing cells, preferably CD25⁺ expressing T cells and more preferably CD25⁺ expressing Treg cells, with a higher efficiency as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention present a lower immunogenicity as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention present a higher stability as compared to the fusion proteins or antibodies of the prior art; in some embodiments, the fusion proteins of the present invention present a better cross-reactivity for the cynomolgus monkey CD25 as compared to the fusion proteins or antibodies of the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0549] Figure l is a histogram showing the binding level of a fusion protein according to the present invention (i.e. ALD2515 V1) to IL-Ra/CD25 protein with or without prior enzymatic digestion, measured in a binding kinetic assay by BioLayer Interferometry (BLI).

[0550] Figure 2 is a histogram showing the binding level of a fusion protein according to the present invention (i.e. ALD2515 V1) to IL-2RP/CD122 protein with or without prior enzymatic digestion, measured in a binding kinetic assay by BioLayer Interferometry (BLI).

[0551] Figure 3 is a graph showing pSTAT5 activation of conventional CD4+ T cells using ALD2515 C1 control construct, ALD2515 V1 and ALD2515 V2 constructs and human recombinant IL-15 as a control, at different concentrations (from 10'¹⁴ to 10'⁸M).

[0552] Figure 4 is a graph showing pSTAT5 activation of CD8+ T cells using ALD2515 C1 control construct, ALD2515 V1 and ALD2515 V2 constructs and human recombinant IL- 15 as a control, at different concentrations (from 10'¹⁴ to 10'⁸M). [0553] Figure 5 is a graph showing pSTAT5 activation of NK cells among PBMCs cells (CD56^bnght NK cells) using ALD2515_C1 control construct, ALD2515_V1 and ALD2515 V2 constructs and human recombinant IL- 15 as a control, at different concentrations (from 10'¹⁴ to 10'⁹M).

[0554] Figure 6 is a histogram showing the IL-15-induced proliferation of CD8+ T cells, measured by the percentage of Far Red cells, using ALD2515 C1 control construct, ALD2515 V1 and ALD2515 V2 constructs and human recombinant IL-15 as a control, at 100 nM.

[0555] Figure 7 is a histogram showing the IL-15-induced proliferation of NK cells (CD56^bnght NK cells), measured by the percentage of Far Red cells, using ALD2515_C1 control construct, ALD2515 V1 and ALD2515 V2 constructs and human recombinant IL- 15 as a control, at 10 nM.

[0556] Figure 8 is a combination of two schemas, showing a control construct comprising a non-masked IL- 15 cytokine linked with an uncleavable linker to an anti- CD25 antibody (Figure 8A) and a construct according to the present invention, comprising a masked IL- 15 cytokine linked to an anti-CD25 antibody, wherein the IL- 15RP masking moiety is linked to the antibody with a cleavable linker (VI and V2 differ by the size of the cleavable linker) (Figure 8B).

[0557] Figure 9 is a graph showing pSTAT5 activation of conventional CD4+ T cells using ALD2515_V1 (Figure 9A) and ALD2515_V2 (Figure 9B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0558] Figure 10 is a graph showing pSTAT5 activation of conventional CD8+ T cells using ALD2515 V1 (Figure 10A) and ALD2515_V2 (Figure 10B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking)..

[0559] Figure 11 is a graph showing pSTAT5 activation of NK cells among PBMCs cells (CD56^brigbtNK cells) using ALD2515_V1 (Figure 11 A) and ALD2515_V2 (Figure 11B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking)..

[0560] Figure 12 is a graph showing pSTAT5 activation of conventional CD4+ T cells using ALD25FO6_V1 (Figure 12A) and ALD25F06_V2 (Figure 12B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking)..

[0561] Figure 13 is a graph showing pSTAT5 activation of conventional CD8+ T cells using ALD25FO6_V1 (Figure 13A) and ALD25F06_V2 (Figure 13B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0562] Figure 14 is a graph showing pSTAT5 activation of NK cells among PBMCs cells (CD56^bright NK cells) using ALD25FO6_V1 (Figure 14A) and ALD25F06_V2 (Figure 14B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0563] Figure 15 is a graph showing pSTAT5 activation of conventional CD4+ T cells using ALD25FO7_V1 (Figure 15A) and ALD25F07_V2 (Figure 15B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0564] Figure 16 is a graph showing pSTAT5 activation of conventional CD8+ T cells using ALD25FO7_V1 (Figure 16A) and ALD25F07_V2 (Figure 16B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0565] Figure 17 is a graph showing pSTAT5 activation of NK cells among PBMCs cells (CD56^bright NK cells) using ALD25FO7_V1 (Figure 17A) and ALD25F07_V2 (Figure 17B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0566] Figure 18 is a graph showing pSTAT5 activation of conventional CD4+ T cells using ALD25F13_V1 (Figure 18A) and ALD25F13_V2 (Figure 18B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0567] Figure 19 is a graph showing pSTAT5 activation of conventional CD8+ T cells using ALD25F13_V1 (Figure 19A) and ALD25F13_V2 (Figure 19B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0568] Figure 20 is a graph showing pSTAT5 activation of NK cells among PBMCs cells (CD56^bright NK cells) using ALD25F13_V1 (Figure 20A) and ALD25F13_V2 (Figure 20B) constructs and human recombinant IL- 15 as a control, at different concentrations, and either before or after MMP8 digestion (i.e. unmasking).

[0569] Figure 21 is a combination of schemas showing five examples of fusion proteins according to the present invention (Constructs A-E). Constructs A, and C comprise an anti-CD25 antibody, a human IL-15 (hIL-15) and an IL-15 binding moiety. Constructs B, D and E further comprise a human IL-15 receptor a sushi domain (hIL-15Ra sushi).

[0570] Figure 22 is a combination of 3 histograms showing the selective depletion of Treg cells (A) but not conventional CD4+ (B) and CD8+ cells (C) from healthy donor’s PBMCs using ALD25F06 V1 and ALD25F06 V2 constructs versus the control anti- CD25 antibody E04-2 (ALD25F06) and an isotype IgGl antibody (huIgGl).

[0571] Figure 23 is a combination of 3 histograms showing the selective depletion of Treg cells (A) but not conventional CD4+ (B) and CD8+ cells (C) from healthy donor’s PBMCs using ALD25F07 V1 and ALD25F07 V2 constructs versus the control anti- CD25 antibody B05 (ALD25F07) and an isotype IgGl antibody (huIgGl).

[0572] Figure 24 is a combination of 3 histograms showing the selective depletion of Treg cells (A) but not conventional CD4+ (B) and CD8+ cells (C) from healthy donor’s PBMCs using ALD25F13 V1 and ALD25F13 V2 constructs versus the control anti- CD25 antibody G02-2 (ALD25F13) and an isotype IgGl antibody (huIgGl). EXAMPLES

[0573] The present invention is further illustrated by the following examples.

Materials and Methods

Constructions

[0574] Several constructs were tested:

A control construct, i.e. ALD2515 C1, which includes a non-masked IL- 15 cytokine linked to the anti-CD25 antibody MAB10 (see Figure 8A), and Several constructs according to the present invention, i.e. ALD2515 V1 and ALD2515 V2, ALD25FO6_V1 and ALD25F06_V2, ALD25FO7_V1 and ALD25F07 V2, ALD25F13_V1 and ALD25F13_V2, which comprise an IL-15 binding moiety (i.e. IL-15RP), an IL- 15 cytokine and an anti-CD25 antibody (i.e. the anti-CD25 antibody MAB10, E04-2, B05 or G02-2, respectively) (see Figure 8B). Constructs VI and V2 only differ by the size of the cleavable linker.

[0575] The amino acid sequences of the constructs are provided hereinbelow:

- ALD2515 C1 with SEQ ID NOs: 130-132,

- ALD2515 V1 with SEQ ID NOs: 133-134 and 132,

- ALD2515 V2 with SEQ ID NOs: 134-135 and 132,

- ALD25F06 V1 with SEQ ID NOs: 136-138,

- ALD25F06 V2 with SEQ ID NOs: 137-139,

- ALD25FO7_V1 with SEQ ID NOs: 140-142,

- ALD25F07 V2 with SEQ ID NOs: 141-143,

- ALD25F13 V1 with SEQ ID NOs: 144-146, and

- ALD25F13 V2 with SEQ ID NO: 145-147.

[0576] DNA coding for the amino acid sequences as defined hereinabove was synthesized and cloned into expression vectors. Constructs were expressed using a CHO- based transient expression system and cell cultures containing the constructs were clarified and then purified by affinity chromatography before final buffer exchange. IL- 15 induced cell activation (pSTAT5 expression)

[0577] The levels of intracellular STAT5 activation induced by IL-15 (which translates in STAT5 phosphorylation [pSTAT5 signal]) was monitored as follows. Frozen human PBMCs were thawed, washed with RPMI medium, seeded into 96-well round-bottom plate and incubated for Ih at 37 °C, 5% CO2. Cells were stimulated with increasing concentrations, from IxlO'⁵ to lxl0²nM, of recombinant human IL-15 or fusion proteins for 30min at 37 °C. Samples were then stained with anti-CD25, anti-CD127, anti-CD8, anti-CD4, anti-CD3 and anti-CD56 labelled antibodies. The fixed and permeabilized samples were then incubated with a pSTAT5-specific antibody for 30min at RT. pSTAT5 signal was then monitored by flow cytometry.

IL-15 induced cell proliferation

[0578] The proliferation of immune cells following treatment with masked fusion proteins was also assessed. Frozen human PBMCs were thawed, washed and incubated with IpM Cell Trace™ Far Red dye in RPMI medium for 20min at 37 °C, 5% CO2. Cells were seeded in RPMI medium and incubated for 2-4h at 37 °C, 5% CO2. Then, cells were stimulated with increasing concentrations, from IxlO'⁵ to lxl0²nM, of recombinant human IL-15 or fusion proteins for 5 days at 37 °C. Samples were stained with anti-CD25, anti-CD127, anti-CD8, anti-CD4, anti-CD3, anti-CD56 and/or anti-TCRgd labelled antibodies. Cell division was monitored by flow cytometry.

Bindins to IL-2Ra/CD25 protein - Kinetic analysis: Anti-Human Fc Biosensors

[0579] Kinetic assays were performed by first capturing IgG using anti-human Fc biosensors. The mAb capture biosensors were then submerged in wells containing a single concentration of CD25 antigen at 50nM (association stage), followed by a dissociation step in running buffer. To allow for reference correction blank sensors were dipped into wells containing the antigen. This referencing provided a means of compensating for non-specific binding of the antigen to the sensor surface. Steps were performed at 25°C at a constant flow-rate of 1000 rpm. New sensors were used for each sample. The response data for the binding of antigen to the surface immobilized IgG were fitted to a 1 : 1 binding model, yielding the data traces. Dissociation rate constants (KD) were calculated using the ForteBio Data Analysis software. Antigen binding (IL- 2Ra/CD25) was determined for pre-digested samples and digested samples.

Binding to IL2-RJ3 protein - Kinetic analysis: Streptavidin Biosensors

[0580] Kinetic assays were performed by first capturing antigen using high precision streptavidin biosensors. Biotinylated human IL2-RP/CD122 was diluted in freshly prepared running buffer. The antigen captured biosensors were then submerged in wells containing a single concentration of antibody, lOOnM (association stage), followed by a dissociation step in running buffer. To allow for reference correction blank sensors were dipped into wells containing the antigen. This referencing provided a means of compensating for non-specific binding of the antigen to the sensor surface. Steps were performed at 25°C at a constant flow-rate of 1000 rpm. New sensors were used for each sample. The response data for the binding of antibody to the surface immobilized antigen were fitted to a 1 : 1 binding model, yielding the data traces. Dissociation rate constants (KD) were calculated using the ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.

Enzymatic Digestion protocol

[0581] MMP proenzymes (pro-MMPs) required activation with APMA for either Ih or 24h at 37°C. Fusion proteins (lOOpg/ml) were incubated with pro-MMPs or activated MMPs (final concentration of 1 pg/ml) at 37°C for Oh, Ih and 24h. Reactions were stopped with the addition of EDTA. Digested samples were buffer-exchanged into PBS using disposable desalting columns prior to any further assay.

Treg depletion

[0582] Autologous TREG depletion assays were performed by incubating control antibodies, undigested masked fusion proteins or unmasked MMP8-digested fusion proteins (Ipg/mL) together with freshly isolated human PBMCs for 4 days at 37 °C, 5% CO2. After incubation, cells were labelled and monitored by flow cytometry for their expression of a panel of cell surface markers including CD45, CD3, CD4, CD8, CD25, CD 127 among others. Results were expressed as the percentage of either TREG cells or conventional CD4+ or CD8+ T cells, among the parental CD45+ cell population.

Results

[0583] As shown in Figure 1, the fusion proteins of the present invention are capable of binding to IL-Ra/CD25 protein in a binding kinetic assay by BioLayer Interferometry (BLI). The measured binding was equivalent with or without prior enzymatic digestion.

[0584] In addition, Figure 2 shows that the binding to IL-2RP/CD122 is strongly increased by the enzymatic digestion of the fusion protein allowing the cleavage of the masking moiety, in a binding kinetic assay by BioLayer Interferometry (BLI).

[0585] The ability of the fusion proteins of the present invention to induce immune cells activation and proliferation was further evaluated.

[0586] First, Figures 3-5 show the results from pSTAT5 activation assays using ALD2515 C1 control construct, ALD2515 V1 and ALD2515 V2 constructs and human recombinant IL- 15 as a control. The ALD2515 C1 includes a non-masked IL- 15 cytokine linked to a CD25 antibody and the ALD2515 V1 and ALD2515 V2 constructs include a masked IL-15 cytokine linked to an anti-CD25 antibody. The levels of STAT5 activation (% percentage of pSTAT5 positive cells) are shown for conventional CD4⁺ T cells (Figure 3), CD8⁺ T cells (Figure 4) and NK cells among PBMCs (Figure 5). In this assay, ALD2515 C1 construct triggers STAT5 activation in sharp contrast with both ALD2515_V1 and ALD2515_V2 masked constructs.

[0587] In addition, the ability of the fusion proteins of the present invention to induce the proliferation of CD8⁺ T cells and NK cells was measured in a Far-red cell proliferation assay, using ALD2515_C1 control construct, ALD2515_V1 and ALD2515_V2 constructs and human recombinant IL- 15 as a control. While ALD2515_C 1 triggers CD8⁺ T cells (Figure 6) and NK cells (Figure 7) proliferation, ALD2515 V1 and ALD2515 V2 masked constructs barely do so. [0588] The same experiments were done with the constructs ALD2515 V1 and ALD2515 V2 masked constructs in presence of the MMP8 protease, which cleaves the cleavable linker between the masking moiety and the anti-CD25 antibody. The levels of STAT5 activation are shown for conventional CD4⁺ T cells (Figures 9A-B), CD8⁺ T cells (Figures 10A-B) and NK cells among PBMCs (Figure 11A-B) for the constructs ALD2515_V 1 and ALD2515_V2, in presence or in absence of the MMP8 protease. These data show that the constructs ALD2515 V1 and ALD2515 V2 trigger STAT5 activation of CD4⁺ T cells, CD8⁺ T cells and NK cells in presence of the MMP8 protease, while the same constructs barely do so in absence of such protease.

[0589] Thus, these data confirm that the fusion proteins of the present invention are inactive when masked, but are able to be activated in presence of proteases. Indeed, the proteases, by cleaving the cleavable linker, can unmask the IL-15, which is then free to bind to its native receptors.

[0590] In addition, the ability of other fusions proteins of the present invention, differing from the constructs ALD2515_V1 and ALD2515_V2 by the anti-CD25 antibody, was evaluated. In particular, the ability of the constructs ALD25F06 V1 and ALD25F06 V2, ALD25FO7_V1 and ALD25F07_V2, and ALD25F13_V1 and ALD25F13_V2, to induce immune cells activation was assessed at different concentrations, in presence or in absence of the MMP8 protease. As shown in Figures 12 to 20, all the constructs ALD25FO6_V1 and ALD25F06_V2, ALD25FO7_V1 and ALD25F07_V2, and ALD25F13 V1 and ALD25F13 V2 are ableto trigger STAT5 activation of CD4⁺ T cells, CD8⁺ T cells and NK cells in presence of the MMP8 protease, while the same constructs barely do so in absence of such protease.

[0591] Altogether, these data show that the fusion proteins of the present invention are able to unmask in presence of proteases, so that IL- 15 is free to bind to its native receptors.

[0592] Finally, the ability of the fusion proteins of the present invention to deplete Treg cells was evaluated using the constructs ALD25FO6_V1, ALD25F06_V2, ALD25FO7_V1, ALD25F07_V2, ALD25F13_V1 and ALD25F13_V2, in presence or in absence of the MMP8 protease. As shown in Figures 22, 23 and 24, the fusion proteins of the present invention, either masked or unmasked, are able to deplete Treg cells, but no conventional CD4+ and CD8+ cells. Interestingly, the fusion proteins of the present invention are as efficient as the anti-CD25 antibodies alone to deplete Treg cells, demonstrating that the anti-CD25 antibodies comprised in the fusion proteins of the present invention retain their full ability to deplete Treg cells.

Claims

1. A fusion protein comprising: i. at least one antibody or antigen-binding fragment thereof, ii. at least one cytokine, iii. at least one cytokine binding moiety capable of binding the at least one cytokine, and iv. at least one cleavable linker, wherein the at least one cleavable linker links the at least one cytokine binding moiety to any one of the elements of the fusion protein, wherein the cleavage of the at least one cleavable linker releases the at least one cytokine binding moiety, thereby leading to the unmasking of the at least one cytokine.

2. The fusion protein according to claim 1, wherein the at least one cleavable linker is cleavable by at least one tumor-associated protease, preferably wherein the at least one tumor-associated protease is selected from the group comprising matrix metallopeptidases (MMPs), cathepsins and urokinase-type Plasminogen Activator (uPA), more preferably the at least one cleavable linker comprises the sequence GGGGSPLGLAGFLGS (SEQ ID NO: 30) or the sequence

GGGGSPLGLAGGGGS(SEQ ID NO: 129).

3. The fusion protein according to any one of claims 1 or 2, wherein the at least one cytokine is an interleukin, preferably an interleukin (IL) selected from the group comprising IL-15, IL-2, IL-4, IL-7, IL-9, IL-12, IL-21, and variants or fragments thereof.

4. The fusion protein according to any one of claims 1 to 3, wherein the at least one cytokine binding moiety comprises or consists of at least one chain of a native receptor of the at least one cytokine, or a fragment or variant thereof.

5. The fusion protein according to claim 4, wherein the at least one cytokine is IL-15 or a fragment or variant thereof, and wherein the at least one cytokine binding moiety comprises or consists of at least one chain of the IL- 15 receptor, or a fragment or variant thereof. The fusion protein according to claim 5, wherein the at least one cytokine moiety comprises or consists of the P chain of the IL-15 receptor, or the domain DI of the P chain of the IL- 15 receptor, or fragments or variants thereof. The fusion protein according to any one of claims 1 to 6, wherein said fusion protein further comprises an IL- 15 receptor a sushi domain or a fragment or variant thereof. The fusion protein according to any one of claims 1 to 7, wherein the fusion protein further comprises at least one uncleavable linker, preferably wherein the at least one uncleavable linker links the at least one cytokine and/or the IL- 15 receptor a sushi domain to any one of the elements of the fusion protein, more preferably the at least one uncleavable linker is a glycine-serine polymer. The fusion protein according to any one of claims 1 to 8, wherein the at least one antibody or antigen-binding fragment thereof specifically binds a marker of immune cells, preferably a marker of immune cells selected from the group comprising CD25, CD160-TM, PD-1, PDL1, CCR8 and CTLA4, more preferably said at least one antibody or antigen-binding fragment thereof specifically binds CD25. The fusion protein according to any one of claims 1 to 9, wherein the at least one antibody or antigen-binding fragment thereof specifically binds CD25, and wherein said at least one antibody or antigen-binding fragment thereof comprises one of the following combinations of complementary-determining regions (CDRs) in the variable regions of the heavy and light chains (VH and VL):

- VH-CDRs 1-3: SEQ ID NO: 1, 11, 19 and VL-CDRs 1-3: SEQ ID NO: 25, 5, 16, respectively

- VH-CDRs 1-3: SEQ ID NO: 1, 11, 12 and VL-CDRs 1-3: SEQ ID NO: 13, 14, 6, respectively - VH-CDRs 1-3: SEQIDNO: 1, 11, 12 and VL-CDRs 1-3: SEQIDNO: 15, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 12 and VL-CDRs 1-3: SEQIDNO: 17, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 12 and VL-CDRs 1-3: SEQIDNO: 18, 5, 6, respectively

- VH-CDRs 1-3: SEQIDNO: 1, 11, 19 and VL-CDRs 1-3: SEQIDNO: 20, 5, 16, respectively

- VH-CDRs 1-3: SEQIDNO: 1,28, 19, and VL-CDRs 1-3: SEQIDNO: 24, 5, 16, respectively - VH-CDRs 1-3: SEQ ID NO: 1, 29, 19, and VL-CDRs 1-3: SEQ ID NO: 25, 5, 6, respectively.

11. A nucleic acid encoding a fusion protein according to any one of claims 1 to 10.

12. An expression vector comprising a nucleic acid according to claim 11. 13. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 10, a nucleic acid according to claim 11, or an expression vector according to claim 12, and a pharmaceutically acceptable excipient.

14. The fusion protein according to any one of claims 1 to 10, the nucleic acid according to claim 11, the expression vector according to claim 12, or the pharmaceutical composition according to claim 13, for use as a medicament.

15. The fusion protein according to any one of claims 1 to 10, the nucleic acid according to claim 11, the expression vector according to claim 12, or the pharmaceutical composition according to claim 13, for use in treating a cancer or an infectious disease in a subject in need thereof.