Molecules comprising masking linkers and uses thereof for the treatment of auto-immune or inflammatory diseases and disorders

Abstract

The present invention relates to a molecule comprising an antigen binding domain derived from an antibody; one or more cytokines having an anti-inflammatory effect covalently linked to the antigen binding domain; and one or two peptide linker(s) covalently linked to the cytokine, the peptide linker(s) being suitable for masking the effect of the cytokine. It also relates to the therapeutic uses of said molecule for the treatment of an auto-immune or inflammatory disease or disorder.

Description

[0001] MOLECULES COMPRISING MASKING LINKERS AND USES THEREOF FOR THE TREATMENT OF AUTOIMMUNE OR INFLAMMATORY DISEASES AND DISORDERS

[0002] FIELD OF THE INVENTION

[0003] The invention pertains to the field of medicine, in particular immunotherapy. It relates to a molecule comprising an antigen binding domain derived from an antibody and a cytokine having an antiinflammatory effect, the molecule comprising linker(s) suitable for masking the effect of the cytokine. It further relates to the use of the molecule for the treatment of auto-immune or inflammatory diseases or disorders.

[0004] BACKGROUND OF THE INVENTION

[0005] Multifunctional molecules are currently the object of developments in immunology, especially in the field of auto-immune or inflammatory diseases or disorders. Such multifunctional molecules typically comprise an antigen binding domain directed to a particular target (e.g., on immune cells) and a cargo (e.g., an immunoactive molecule such as a cytokine). Even though they present many advantages, these molecules may also present inconveniences. The design of multifunctional molecules implies several key attributes such as binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, compatibility with the attachment of additional domains but also production yield and cost compatible with a clinical development.

[0006] For instance, bifunctional molecules based on an antibody linked to various interleukins, or other immunotherapeutic agents have been disclosed, in particular in WO2018 / 184964 or W02018071919. Such molecules provide the advantages to target tissues thanks to the antibody interaction, and in the same time allow to deliver interleukins into the targeted site. However, optimal targeting remains difficult to achieve. To circumvent this inconvenient, different solutions have been proposed.

[0007] A first strategy is based on the masking of the immunotherapeutic agents by a molecule, protein or peptide, such as antigen binding domains or receptors, that have a binding affinity for the immunotherapeutic agents. However, this strategy comprises the inclusion of additional moieties in the multifunctional molecule and the risks of insufficient demasking once onsite. Additional moieties increase the size and weight of the multifunctional molecule, affecting its pharmacokinetic characteristics and distribution, which in turn may render the multifunctional molecule less effective than a standard medication. Circumventing this issue with a double binding antibody is complicated as it needs to search, find and test multiple potential antigen binding domain for each pair of target and immunotherapeutic agent to assess efficiency and safety.

[0008] Other strategies are based on cleavable linkers, i.e., linkers which mask the cargo of bifunctional molecules until its arrival at a delivery site. Once onsite, the linker is cleaved to release its cargo. Multiple linkers with physicochemical properties were used to cleave cytokine by proteases into targeted sites and to release cytokines specifically into targeted environments (Hsu et al, 2021, Nat. Commun., 12, 2768; Cao et al, 2021, Nat. Commun., 12, 5866 ; Desnoyers et al, 2013, Sci. Transl. Med., 5). This strategy improves cytokine biodisponibility and prevents off-target in periphery. Protease activable masked cytokines have been reported by using a variety of masking moieties.

[0009] Nevertheless, this strategy still comprises the risks of insufficient activation in the targeted environment and of undesired activation in non-targeted tissues. Indeed, this solution has the inconvenient to be dependent to the type and quantity of proteases present in the targeted microenvironment for its efficacy.

[0010] There remains therefore a significant need in the art for new and improved multifunctional molecule for safe immunotherapy, notably against auto-immune or inflammatory diseases or disorders. The present inventors have made a significant step forward with the invention disclosed herein.

[0011] SUMMARY OF THE INVENTION

[0012] The applicant has developed an innovative solution allowing to control the activity of any immunotherapeutic molecule by masking peptide linkers that are not cleavable, and thereby provides a reliable platform usable with a large array of immunotherapeutic molecules with minimal to no modification whatsoever.

[0013] The advantage of the linker peptides according to the invention is their ability to mask a cytokine, in particular its activity and / or binding to its cognate receptor. In the multifunctional molecules of the invention, it is the binding of the targeting moiety (e.g., antibody) to its target that enables unmasking / demasking of the cytokine and its binding to its receptor. These masking and demasking properties are intrinsic to the composition / sequence of the linkers of the invention. This is why the inventor have thought of the name "cytomask" or "cytomasking" linkers when referring to the linkers of the invention.

[0014] Such platform allows to include molecules having an effect too important to be safely used alone. For example, wild type cytokines such as IL-10 can be used in the multifunctional molecule of the invention, without further modification, even though their use is usually discouraged because of their pleiotropic side effect.

[0015] It is especially efficient to target site of inflammation, by selecting an antigen binding domain (ABD) which targets activated immune cells, especially activated immune cells located in the site of inflammation. For example, a multifunctional molecule of the invention comprising an anti-SIRPalpha binding domain bearing a cytokine having an anti-inflammatory effect will only be activated in the inflammation microenvironment. If, for any reason, the molecule was to exit the tumoral microenvironment to return to the blood flow or to access healthy tissue, the lack of SIPRalpha positive cells would trigger a switch of the multifunctional molecule and masking the cytokine until the recognition of a SIPRalpha positive cell. Moreover, the greatest advantage of using non-cleavable linkers is their increased plasma stability when compared to many cleavable linkers. Despite the limited "bystander" effect, the resistance to cleavage outside of targeted cells may actually increase the specificity of drug release. For example, several in vivo studies and clinical data have shown that non-cleavable linked ADCs outperform their cleavable counterparts (indicated in Combs et al, AAPS J. 2015 Mar;17(2):339-51). It is thus believed that the peptide linkers provided herein will increase therapeutic efficiency of multifunctional molecules.

[0016] This solution leads to a multifunctional molecule which gathers the advantages of both cleavable and non-cleavable linkers: great plasma stability, excellent specificity to a specific environment and independence from proteases to uncover the anti-inflammatory cytokine when the multifunctional molecule is onsite.

[0017] Another major advantage of the molecule of the invention is its capacity to act via cis-targeting (i.e., on the same cell), and exert a synergistic effect on the targeted cells. Cleavable linkers, by releasing the cytokine in the media, are incapable of engaging an additional target.

[0018] The multifunctional molecules of the invention have particularly advantageous and surprising properties: cis-masking of the anti-inflammatory cytokine until the recognition of the multifunctional molecules to the targeted cells (interaction between the antigen binding domain of the multifunctional molecule with a targeted antigen on the targeted cell); cis-demasking of said anti-inflammatory cytokine consecutively to said recognition, without uncontrolled liberation and / or loss of efficiency; the consecutive cis-activation of the target cell with synergy of i) the effect of the ABD and ii) the effect of the anti-inflammatory cytokine, for instance the synergistic activation of a myeloid cell by both i) SIRPalpha (via the antigen binding domain of the multifunctional molecule) and ii) a cytokine (such as IL-10).

[0019] The absence of trans-activity is yet another advantage of the invention, as it limits or suppress the possibility of a binding and activation of cells out of the desired environment, thus limiting the risk of an off-target effect and possible toxicity.

[0020] Compared to some molecule of the prior art, the multifunctional molecule of the invention also features a pharmacokinetic profile similar to unmasked multifunctional molecules, hence avoiding any risk of poor distribution or toxicity.

[0021] The invention relates to a multifunctional molecule comprising or consisting essentially of: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having an anti-inflammatory effect covalently linked to the N-terminal and / or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker covalently linked to the N-terminal and / or C-terminal end of the cytokine; wherein the peptide linker is 5 to 30 amino acids in length and consists of

[0022] - at least 20% acidic or amidic amino acids independently selected from the group consisting of E, D and N,

[0023] - up to 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and

[0024] - at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.

[0025] Typically, the peptide linker is a masking linker .

[0026] Optionally, the peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; a sequence having 5 to 30 consecutive amino acids N.

[0027] Optionally, said peptide linker comprises, essentially consists of or consists of:

[0028] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids selected independently from the group consisting of E, D and N; or

[0029] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being E; or

[0030] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being D; or

[0031] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being N.

[0032] Optionally, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),

[0033] NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),

[0034] DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),

[0035] NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).

[0036] Optionally, the peptide linker is covalently linked to the N-terminal end of the cytokine and is connecting the antigen binding domain and the cytokine.

[0037] Said peptide linker is called the "masking" peptide linker.

[0038] Optionally, the multifunctional molecule comprises two peptide linkers, a first peptide linker being as described herein (i.e., a "masking peptide linker) and a second peptide linker, wherein:

[0039] - the second peptide linker has an amino acid sequence as defined for the first linker (i.e., a "masking peptide linker); or

[0040] - the second peptide linker is identical to the first peptide linker; or

[0041] - the second peptide linker is between 10 and 30 amino acids in length and consists of less than from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.

[0042] Optionally, the first peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the second peptide linker is linked to the C-terminal end of the cytokine. Alternatively, the second peptide linker is covalently linked to the N- terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the first peptide linker is linked to the C-terminal end of the cytokine.

[0043] Preferably, the multifunctional molecule comprises a first peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5) and a second peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5), preferably wherein:

[0044] - the N-terminal end of the first peptide linker is covalently linked to the C-terminal end of the antigen binding domain, preferably in C-terminal end of a Fc domain;

[0045] - the C-terminal end of the first peptide linker is covalently linked to the N-terminal end of the cytokine; and

[0046] - the N-terminal end of the second peptide linker is covalently linked to the C-terminal end of the cytokine.

[0047] Optionally, the multifunctional molecule comprises a single antigen binding domain, optionally a Fc domain, and a single cytokine.

[0048] Optionally, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of antigen binding domain and of cytokine; and b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) either

[0049] - the first peptide linker and the single cytokine, variant or fragment thereof; said first peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine; or

[0050] - the first and second peptide linkers and the single cytokine, variant or fragment thereof; wherein the first peptide linker is covalently linked to the N-terminal end of the cytokine and the second peptide linker is linked to the C-terminal end of the cytokine.

[0051] Optionally, the anti-inflammatory cytokine, variant or fragment thereof is a cytokine selected from the group consisting of TGFP, IL-2, IL-4, IL-10, IL-11, IL-12, IL-13, IL-14, IL-19, IL-22, IL-24, IL-25, IL-27, IL-35, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine, preferably a cytokine selected from the group consisting of IL-2, IL-10, IL-35, IL-37 and IL-38 or a variant thereof, even more preferably IL-2 or IL-10 or a variant thereof. Optionally, the antigen binding domain binds to a target expressed on immune cells surface and is preferably selected from the group consisting of CD3, CD4, CD8, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD119, CD137 / 4-1BB / TNFRSF9, CD150 / SLAMF1, CD153, CD154 / CD40L, CD28, CD223 / LAG-3, CD226, CD25, CD254, CD26, CD27, CD30, CD39 / ENTPD1, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, LFA-1, TIM-1, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE / CD103, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PD-1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206 / MRC1, LTBR / CD70, TNFSF14, SLAMF7, NKG2A, 2B4, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270,

[0052] GITR / TNFRSF18 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD74, CD165, 4-1BBL / CD137L, ICOSL / CD275, SIRPa, SIRPG, TREM2, LILRB2, Dectin-1, CLEVER1 and CD160, more preferably selected from the group consisting of SIRPa, TREM2, LILRB2, CD206 / MRC1, CD163, CLEC-1A, Dectin-1, CLEVER1 and CLEC-9A.

[0053] In some aspects, the antigen binding domain is a humanized anti-human SIPRalpha antibody or antigen-binding fragment thereof having an agonist effect on SIRPalpha.

[0054] Alternatively, the antigen binding domain is a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, preferably comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 74 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 75; ii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 96 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 97; and iii) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 104 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 105.

[0055] Alternatively, the antigen binding domain is an anti-human TIGIT antibody or antigen-binding fragment thereof, preferably comprising or consisting of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 133 and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 134. The invention also relates to an isolated nucleic acid molecule, a group of isolated nucleic acid molecules or a vector encoding the multifunctional molecule as described herein. The invention also relates to a host cell comprising the isolated nucleic acid molecule and / or the group of isolated nucleic acid molecules and / or the vector as described herein.

[0056] The invention also relates to a pharmaceutical composition comprising a multifunctional molecule as described herein.

[0057] The invention also relates to the multifunctional molecule or the pharmaceutical composition as described herein for use as a medicament, preferably in the treatment of an auto-immune or inflammatory disease or disorder. It also relates to the use of the multifunctional molecule or the pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of an auto-immune or inflammatory disease or disorder. It further relates to a method for treating an auto-immune or inflammatory disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the multifunctional molecule or the pharmaceutical composition as described herein to said subject.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] Definitions

[0060] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.

[0061] As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2) or subclass. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Unless specifically noted otherwise, the term "antibody" includes intact immunoglobulins and "antibody fragment" or "antigen binding fragment" (such as Fab, Fab', F(ab')2, Fv), single chain (scFv or scFab), CrossMAb, mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to a humanized antibody. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CHI" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341- 447 according to the EU index as in Kabat.

[0062] An "antibody heavy chain" as used herein, refers to the larger of the two types of polypeptide chains present in antibody conformations. The CDRs of the antibody heavy chain are typically referred to as "HCDR1", "HCDR2" and "HCDR3". The framework regions of the antibody heavy chain are typically referred to as "HFR1", "HFR2", "HFR3" and "HFR4". An antibody heavy chain is typically structured as follows : HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0063] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations; K and X light chains refer to the two major antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as "LCDR1", "LCDR2" and "LCDR3". The framework regions of the antibody light chain are typically referred to as "LFR1", "LFR2", "LFR3" and "LFR4". An antibody light chain is typically structured as follows : LFR1-LCDR1-LFR2-LCDR2- LFR3-LCDR3-LFR4.

[0064] By "amino acid change" or "amino acid modification" is meant herein a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitution, insertion and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" are used interchangeably and refer to the position of a particular amino acid in an amino acids sequence, generally specified with the one letter codes for the amino acids. The first amino acid in the amino acids sequence (i.e., starting from the N terminus) should be considered as having position 1.

[0065] A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain ("R-group") with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:

[0066] Table A - Amino Acid Residue

[0067] Table B - Alternative Conservative Amino Acid Residue Substitution Groups Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues

[0068] As used herein, the "sequence identity" between two sequences is described by the parameter "sequence identity", "sequence similarity" or "sequence homology". For purposes of the present invention, the "percentage identity" between two sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods in the art, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool "Emboss needle" for pairwise sequence alignment of proteins providing by EMBL-EBI and available on: www.ebi.ac. uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, for example using default settings: (I) Matrix : BLOSUM62, (ii) Gap open : 10, (iii) gap extend : 0.5, (iv) output format : pair, (v) end gap penalty : false, (vi) end gap open : 10, (vii) end gap extend : 0.5.

[0069] Alternatively, Sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with the default settings.

[0070] The terms "derive from" and "derived from" as used herein refers to a compound having a structure derived from the structure of a parent compound or protein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar properties, activities and utilities as the claimed compounds.

[0071] As used herein, the terms "disorder" or "disease" refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease e.g., an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to auto-immune and / or inflammatory diseases or disorders that affect animals and / or humans.

[0072] The term "agonist" as used herein, refers to a substance that activates the functionality of an activating receptor. Particularly, this term refers to an antibody that binds to a cellular activating receptor as a reference substance, and has at least partially the same effect of the biologically natural ligand (e.g., inducing the activator effect of the receptor).

[0073] The term "antagonist" as used herein, refers to a substance that blocks or reduces the activity or functionality of another substance. Particularly, this term refers to an antibody that binds to a receptor or protein as a reference substance, and have at least partially the same effect of the biologically natural ligand (e.g., inducing the inhibition of the receptor). Preferably, this term refers to an antibody that binds to a cellular receptor as a reference substance (e.g. biologically natural ligand of the receptor), preventing it from producing all or part of its usual biological effects (e.g., thereby inhibiting the effect of the receptor when it is bound by its biologically natural ligand).

[0074] As used herein, "trans-targeting" and "trans-activity" refer to the capacity of a multifunctional molecule to bond to at least two different targets expressed on different cells and / or cell types.

[0075] As used herein, "cis-targeting" and "cis-activity" refer to the capacity of a multifunctional molecule to bond to at least two different targets expressed on the same cell.

[0076] "Pharmacokinetics" (PK) refers to the movement of drugs through the body, whereas pharmacodynamics (PD) refers to the body's biological response to drugs. PK describes a drug's exposure by characterizing absorption, distribution, bioavailability, metabolism, and excretion as a function of time. PD describes drug response in terms of biochemical or molecular interactions. PK and PD Analyses are used to characterize drug exposure, predict and assess changes in dosage, estimate rate of elimination and rate of absorption, assess relative bioavailability / bioequivalence of a formulation, characterize intra- and inter-subject variability, understand concentration-effect relationships, and establish safety margins and efficacy characteristics. By "improving PK" it is meant that one of the above characteristics is improved, for example, such as an increased half-life of the molecule, in particular a longer serum half-life of the molecule when injected to a subject.

[0077] As used herein, the terms "pharmacokinetics" and "PK" are used interchangeably and refer to the fate of compounds, substances or drugs administered to a living organism. Pharmacokinetics particularly comprise the ADME or LADME scheme, which stands for Liberation (i.e., the release of a substance from a composition), Absorption (i.e., the entrance of the substance in blood circulation), Distribution (i.e., dispersion or dissemination of the substance through the body) Metabolism (i.e., transformation or degradation of the substance) and Excretion (i.e., the removal or clearance of the substance from the organism). The two phases of metabolism and excretion can also be grouped together under the title elimination. Different pharmacokinetics parameters can be monitored by the man skilled in the art, such as elimination half-life, elimination constant rate, clearance (i.e., the volume of plasma cleared of the drug per unit time), Cmax (Maximum serum concentration), and Drug exposure (determined by Area under the curve, see Scheff et al, Pharm Res. 2011 May;28(5):1081-9) among others.

[0078] As used herein, the term "isolated" indicates that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an "isolated" antibody is one which has been identified and separated and / or recovered from a component of its natural environment.

[0079] The term "and / or" as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually. The term "a" or "an" can refer to one of or a plurality of the elements it modifies (e.g., "a reagent" can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.

[0080] The term "about" as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to + / - 10% (e.g., + / - 0.5%, + / -1 %, + / -1 .5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / -9.5%). The use of the term "about" at the beginning of a string of values modifies each of the values (i.e., "about 1, 2 and 3" refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). As used herein, the term "consist essentially of" refers to those elements required for a given embodiment. This term indicates the inclusion of any recited characteristics and permits the optional presence of elements that do not materially affect nor change the characteristics or functions of said embodiment. Preferably, in the context of a multifunctional molecule, it refers to a molecule that comprises the recited elements (e.g., antigen binding domain(s), immunoactive molecule(s) and peptide linker(s)), and optionally includes other elements that do not particular interfere with the structure or function of the molecule, such as peptide spacers.

[0081] The term "at least one" means "one or more" or "one or several". For instance, it refers to one, two, three or more.

[0082] Multifunctional molecule structure

[0083] The multifunctional molecules envisioned herein comprise or consist of one or more antigen binding domain(s), one or more cytokine(s) having an anti-inflammatory effect and one or more peptide linker(s).

[0084] As used herein, the terms "multifunctional molecule" or "polyfunctional molecule" are interchangeable and refer to hybrid or conjugated drugs or as chimeric drugs from two or more drugs having different pharmacological activities. In particular, the multifunctional molecule can be a bifunctional molecule, in which a first function is hold by the anti-inflammatory cytokine(s) and a second function is hold by the antigen binding domain(s) derived from an antibody. However, optionally, the multifunctional molecule can present additional functions such as another binding moiety, another immunoactive molecule or any other active function such as a cytotoxic agent, an antiproliferative agent or an anti-inflammatory agent that have additional or different pharmacological activities.

[0085] The multifunctional molecule of the invention does not comprise a moiety able to bind the cytokine. Preferably, the multifunctional molecule does not comprise a moiety that presents a binding affinity to the cytokine, such as an antibody-antigen or receptor-ligand interaction. Such moiety can be selected from the group consisting of molecules, peptides, proteins, antibodies, antigen binding domain, receptors and fragment thereof that have a binding affinity for the cytokine. Typically, the multifunctional molecule of the invention does not comprise an antigen binding domain that is able to bind to the cytokine comprised in the multifunctional molecule, especially such as a Fab, a scFv or a VHH. For example, when the multifunctional molecule of the invention comprises IL-2, the multifunctional molecule does not comprise an antigen binding domain, such as an scFv, that is able to bind / recognize IL-2. Indeed, in the multifunctional molecule of the invention, it is particularly the peptide linker that is responsible for the masking / demasking of the cytokine. Typically, the multifunctional molecule does not comprise an additional masking moiety or domain (such as an antigen binding domain able to bind the cytokine comprised in the multifunctional molecule). Preferably, the multifunctional molecule does not comprise a moiety that present a binding affinity equal or lower than IO-6M, IO-7M, 10'8M, 10'9M or IO10M for the cytokine. For example, when the cytokine is IL-2, the multifunctional molecule does not comprise an anti-IL2 antigen binding domain.

[0086] Alternatively, or additionally, the multifunctional molecule does not comprise a receptor or a fragment thereof that is able to interact with the cytokine. For example, when the cytokine is IL-2, the multifunctional molecule does not comprise IL-2R or a fragment thereof able to bind IL-2.

[0087] The multifunctional molecule comprises or consists essentially of: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant or fragment thereof having an anti-inflammatory effect covalently linked to the N-terminal and / or C-terminal end of the antigen binding domain; and a non-cleavable peptide linker (i.e., a "masking" peptide linker) covalently linked to the N- terminal and / or C-terminal end of the cytokine; wherein the peptide linker is 5 to 30 amino acids in length and consists of

[0088] - at least 20% acidic or amidic amino acids independently selected from the group consisting of E, D and N,

[0089] - up to 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and

[0090] - at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.

[0091] In some aspects, the multifunctional molecule may comprise or essentially consist of: one or more antigen binding domain(s), one or more cytokine(s), variant(s) or fragment(s) thereof having an anti-inflammatory effect covalently linked to the N-terminal end and / or C-terminal end of the antigen binding domain(s), and one or more peptide linker(s) covalently linked to the N-terminal end and / or C-terminal end of the cytokine(s). As further details herein, the peptide linkers can be identical or different but at least one peptide linker is a "masking" peptide linker as defined above with the ratio of amino acids (i.e., at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).

[0092] More particularly, the multifunctional molecule comprises at least one cytokine, variant or fragment thereof having an anti-inflammatory effect covalently linked to at least one antigen binding domain, wherein one or two peptide linker(s) are covalently linked to the cytokine. When two peptide linkers are present, the linkers are linked to the cytokine so as the latter is flanked by a first linker at one end and by a second linker at the other end and at least one of them is a "masking" peptide linker.

[0093] The multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain(s), one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and / or C-terminal end of the antigen binding domain(s) or the Fc domain, and one to eight peptide linker(s) covalently linked to the N-terminal end and / or C-terminal end of the cytokine(s), variant(s) or fragment(s) thereof, wherein each cytokine is linked to at least one peptide linker as defined above as a "masking" peptide linker (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).

[0094] Preferably, the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain(s), one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and / or C-terminal end of the antigen binding domain(s) or the Fc domain, and two to eight peptide linker(s), wherein a first linker is covalently linked to the N-terminal end of the cytokine(s), variant(s) or fragment(s) thereof and a second linker is linked to the C-terminal end of the cytokine(s), variant(s) or fragment(s) thereof (i.e., two peptide linkers per cytokine) but with at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).

[0095] In particular, the multifunctional molecule may comprise an IgG antibody linked to one, two, three or four cytokine(s), variant(s) or fragment(s) thereof. In a multifunctional molecule, the cytokine(s) can be the same or different. Preferably, the C-terminal end of the IgG antibody chain are covalently linked to the cytokine, preferably to the N-terminal end of the cytokine, preferably through a peptide linker. Such multifunctional molecule particularly comprises:

[0096] - one "masking" peptide linker as defined herein covalently linked to N-terminus or C-terminus of each of the cytokines; or

[0097] - a first peptide linker covalently linked to N-terminus of each of the cytokines and a second peptide linker linked to the C-terminus of each of the cytokines, said peptide linkers being the same or different and with at least one peptide linker being a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).

[0098] In a particular aspect, when the multifunctional molecule comprises an antigen binding domain which is an antibody, in particular an IgG antibody, said antibody is covalently linked to a cytokine, variant or fragment thereof at the C-terminal end of one light chain, at the C-terminal end of both light chains, at the C-terminal end of one heavy chain, at the C-terminal end of both heavy chains, at the C-terminal end of one light chain and at the C-terminal end of one heavy chain, at the C-terminal end of one light chain and at the C-terminal end of both heavy chains, at the C-terminal end of both light chains and at the C-terminal end of one heavy chain, or at the C-terminal end of both light chains and at the C- terminal end of both heavy chains of said antibody.

[0099] Preferably, the cytokine is covalently linked to the antibody at the C-terminal end of one or both heavy chains. Such antibody may comprise heterodimeric Fc domains which promote heterodimeric formation and / or facilitate purification of heterodimers over the homodimers.

[0100] Alternatively, the multifunctional molecule may comprise a diabody (comprising two chains including each a VH domain and a VL domain of an antibody) linked to one or two cytokine(s), variant(s) or fragment(s) thereof. For instance, the multifunctional molecule may comprise a diabody in which each of the two chains are linked to a cytokine or a variant or fragment thereof (i.e., the multifunctional molecule comprises two cytokines, said cytokines being the same or different). Preferably, the C- terminal end of the diabody's chain is covalently linked to the N-terminal end of a cytokine, preferably by a peptide spacer or a peptide linker of the invention. Such multifunctional molecule may comprise:

[0101] - one peptide linker covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end; or - one peptide linker covalently linked to the C-terminus of the cytokine; or

[0102] - one peptide linker covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end, and another peptide linker linked to the C-terminus of the cytokine, said peptide linkers being the same or different; or

[0103] - one peptide spacer covalently linked to the C-terminus of the diabody's chain at one end and to the N-terminus of the cytokine at the other end, and one peptide linker linked to the C-terminus of the cytokine; wherein at least one peptide linker is a "masking peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).

[0104] Alternatively, the multifunctional molecule may comprise a diabody in which only one of the two chains is linked to the cytokine, preferably by a peptide linker or spacer. In another alternative, the cytokine can be linked to two diabodies, said diabodies can be the same or different and being placed either on each side of the cytokine or both at one side.

[0105] In another aspect, the multifunctional molecule comprises only one antigen binding domain, such as a Fab, a scFv or a VHH. In a particular aspect, the multifunctional molecule comprises only one cytokine, variant or fragment thereof. Optionally, the multifunctional molecule comprises only one antigen binding domain and only one cytokine, variant or fragment thereof.

[0106] The multifunctional molecule may particularly comprise, essentially consist or consist of: a single antigen binding domain, optionally a Fc domain linked to the C terminal end of the antigen binding domain, a single cytokine, variant or fragment thereof covalently linked to N-terminus and / or C- terminus to the antigen binding domain or Fc domain, and i) one peptide linker covalently linked to the N-terminal end or C-terminal end of the cytokine; or ii) two peptide linkers; wherein a first linker is covalently linked to the N-terminal end of the cytokine and a second linker is linked to the C-terminal end of the cytokine, wherein at least one peptide linker is a "masking" peptide linker as defined herein (i.e., with at least 20% acidic or amidic amino acids E, D and N, up to 20% basic amino acids H, K and R, and at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T).

[0107] In a particular aspect, the multifunctional molecule is a polypeptide or protein produced as a recombinant protein. Accordingly, the multifunctional molecule is a fusion protein. In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a "masking" peptide linker as defined herein and a cytokine or a variant or fragment thereof.

[0108] In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a cytokine or a variant or fragment thereof and a "masking" peptide linker as defined herein .

[0109] In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a first peptide linker, a cytokine or a variant or fragment thereof and a second peptide linker, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein.

[0110] In a particular aspect, the multifunctional molecule, from the N-terminal to the C-terminal end, comprises or essentially consists of an antigen binding domain or a part thereof, optionally a peptide spacer, optionally a Fc domain, a peptide spacer, a cytokine or a variant or fragment thereof and a "masking" peptide linker as defined herein.

[0111] In a particular aspect, when the multifunctional molecule comprises a single antigen binding domain, the multifunctional molecule comprises an antigen binding domain covalently linked, optionally through a peptide spacer, by its C-terminal end to N-terminal end of a first Fc chain, a complementary second Fc chain forming with the first Fc chain a Fc domain, said first Fc chain being covalently linked by its C-terminal end to the cytokine, variant or fragment thereof and a "masking" peptide linker as defined herein being either between the first Fc chain and the cytokine, variant or fragment thereof or at the C-terminal end of the cytokine, variant or fragment thereof. Optionally, the complementary second Fc chain is devoid of binding domain, devoid of cytokine or devoid of a binding domain and of a cytokine. Preferably, the antigen binding domain is a Fab domain, a Fab', a single-chain variable fragment (scFV) or a single domain antibody (sdAb).

[0112] Such multifunctional molecule comprises:

[0113] - one "masking" peptide linker as defined herein covalently linked to N-terminus or C-terminus of the cytokine; or

[0114] - one peptide linker covalently linked to N-terminus of the cytokine and another peptide linker linked to the C-terminus of the cytokine, said peptide linkers being the same or different and at least one of them being a "masking" peptide linker as defined herein; or

[0115] - one peptide spacer covalently linked to N-terminus of the cytokine and a "masking" peptide linker as defined herein linked to the C-terminus of the cytokine. Preferably, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv)

[0116] - one "masking" peptide linker as defined herein and a single cytokine, variant or fragment thereof; said peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine; or

[0117] - two peptide linkers and a single cytokine, variant or fragment thereof; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein, optionally both; or

[0118] - one "masking" peptide linker as defined herein , one peptide spacer and a single cytokine, variant or fragment thereof; wherein a peptide spacer is covalently linked to the N-terminus of the cytokine and a peptide linker is linked to the C-terminus of the cytokine.

[0119] Preferably, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) two peptide linkers and a single cytokine, variant or fragment thereof; wherein a first linker is covalently linked to the C-terminus of the second Fc chain and to the N- terminus of the cytokine and a second linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers being a "masking" peptide linker as defined herein, optionally both.

[0120] The term "entity" envisioned herein refers to part or moiety of the multifunctional molecule.

[0121] In a particular aspect, when the multifunctional molecule comprises a single antigen binding domain, the multifunctional molecule comprises an antigen binding domain which is a single-chain variable fragment (scFV), covalently linked, optionally through a peptide spacer, by its C-terminal end to N- terminal end of a first Fc chain, a complementary second Fc chain forming with the first Fc chain a Fc domain, said first Fc chain being covalently linked by its C-terminal end to the cytokine, variant or fragment thereof and a "masking" peptide linker as defined herein being either between the first Fc chain and the cytokine, variant or fragment thereof or at the C-terminal end of the cytokine, variant or fragment thereof. Optionally, the complementary second Fc chain is i) devoid of binding domain, ii) devoid of cytokine or iii) devoid of a binding domain and of a cytokine. Preferably, the multifunctional molecule comprises or consists of: a) an antigen binding domain which is a single-chain variable fragment (scFV), b) optionally a Fc domain; and c) two peptide linkers and a single cytokine, variant or fragment thereof; wherein a first linker is covalently linked to the C-terminus of the single-chain variable fragment and to the N-terminus of the cytokine and a second linker is linked to the C-terminus of the cytokine, and wherein at least one of the two peptide linkers is a "masking" peptide linker as defined herein, optionally both.

[0122] In a specific aspect of the invention, the multifunctional molecules envisioned herein comprise or consist of one or more antigen binding domain(s), one or more cytokine(s), one or more "masking" peptide linker(s) as defined herein and one or more additional peptide linker(s).

[0123] In some aspects, the multifunctional molecule may comprise or consist of: one or more antigen binding domain(s), one or more cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and / or C-terminal end of the antigen binding domain, optionally through a peptide spacer, one or more "masking" peptide linker(s) as defined herein covalently linked to the N-terminal end and / or C-terminal end of the cytokine, and one or more additional peptide linker(s) covalently linked to the N-terminal end and / or C- terminal end of the cytokine.

[0124] Particularly, the multifunctional molecule comprises at least one cytokine, variant or fragment thereof covalently linked to at least one antigen binding domain, wherein one peptide linker is covalently linked to the cytokine and one additional peptide linker is covalently linked to the cytokine, wherein at least one of the peptide linkers is a "masking" peptide linker as defined herein, optionally both. When two peptide linkers are present, the linkers are linked to the cytokine so as the latter is flanked by a first linker (the peptide linker or the additional peptide linker) at one end and by a second linker (the additional peptide linker or the peptide linker, respectively) at the other end.

[0125] Preferably, the multifunctional molecule may particularly comprise, essentially consist or consist of: one or two antigen binding domain(s), optionally a Fc domain linked to the C terminal end of the antigen binding domain, one to four cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal end and / or C-terminal end of the antigen binding domain or the Fc domain, and one to four peptide linker(s), covalently linked to the N-terminal end and / or C-terminal end of the cytokine, and one to four additional peptide linker(s), covalently linked to the N-terminal end and / or C- terminal end of the cytokine, wherein a first linker (peptide linker or additional peptide linker) is covalently linked to the N-terminal end of the cytokine and a second linker (additional peptide linker or peptide linker, respectively) is linked to the C-terminal end of the cytokine (i.e., one peptide linker and one additional peptide linker per cytokine) and wherein at least one peptide linker is a "masking" peptide linker as defined herein.

[0126] Examples of the multifunctional molecules are particularly provided herein below under the section "Examples of multifunctional molecules".

[0127] Examples of each element (i.e., antigen binding domain, Fc domain, cytokine and peptide linker, especially "masking" peptide linker as defined herein) comprised in the multifunctional molecules according to the invention are described herein below in their respective sections and apply to any of the multifunctional molecules disclosed herein. Some general illustrative and non-limiting structures are disclosed in Figure 1C.

[0128] In some aspects, the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures Cl, C2, C3, C4, C5, C6, C7 and C8 of Figure 1C.

[0129] Peptide Linkers

[0130] The multifunctional molecule envisioned herein comprises one or more peptide linker(s) and at least one "masking" peptide linker as defined herein. The peptide linker is linked to N-terminus and / or C- terminus of the cytokine.

[0131] By "masking peptide linker" it is meant refers to a short peptide sequence that is strategically designed and incorporated into a larger protein molecule such as an antibody or multifunctional molecule disclosed herein to hide or mask specific functional proteins or epitopes such as a cytokine. Masking peptide linker generally allow avoiding immunogenicity, controlling activation and / or stabilization of the masked proteins or epitopes. In the context of the invention, the masking peptide linker allows the masking or inactivation of the cytokine until the antigen binding domain binds to its target.

[0132] In a multifunctional molecule of the invention, the "masking peptide linker" is typically able to mask the cytokine or the cytokine activity and / or is able to unmask or demask the cytokine or cytokine activity, especially when the antigen biding domain of the multifunctional molecule binds to its target. Typically, the peptide linker of the invention allows to mask and / or unmask or demask the cytokine, preferably the cytokine activity, even more preferably the binding of the cytokine to its receptor.

[0133] By "covalently linked" or "covalently bound", it is meant a chemical bond that involves the sharing of electrons to form electron pairs between atoms between two moieties, e.g., a cytokine and a peptide linker. In the context of the invention, covalent links are not reversible. Preferably, the covalent links are amide bond.

[0134] The "masking" peptide linker is not cleavable or does not contain any protease cleavage site. As used herein, the term "non-cleavable peptide linker" or "uncleavable linker" refers to an amino acid sequence that is designed to remain intact without being cleaved or broken down under certain conditions, such as physiological conditions (e.g. pH), enzymatic or chemical cleavage. Such peptide linkers typically ensure that the linked molecules (e.g., an antigen binding domain and a cytokine such as disclosed herein) remain connected throughout the intended application, whether it is in vivo or in vitro. Preferably, non-cleavable peptide linkers do not comprise a metalloprotease cleavage site. The masking peptide linker particularly does not specifically bind to or recognize the cytokine to which it is covalently linked.

[0135] In the multifunctional molecule of the invention, the number of peptide linker(s) may vary depending on the number of cytokine(s). The molecule comprises one or two peptide linkers per cytokine, but at least one "masking" peptide linker as defined herein. Optionally, the molecule comprises two "masking" peptide linkers as defined herein per cytokine.

[0136] Preferably, the multifunctional molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 peptide linker(s). In particular, the multifunctional molecule may comprise one or two peptide linker(s) if the multifunctional molecule comprises one cytokine, and at least one "masking" peptide linker as defined herein.

[0137] The multifunctional molecule may comprise two to four peptide linkers if the multifunctional molecule comprises two cytokines, and at least two "masking" peptide linkers as defined herein.

[0138] The multifunctional molecule may comprise three to six peptide linkers if the multifunctional molecule comprises three cytokines, and at least three "masking" peptide linkers as defined herein.

[0139] The multifunctional molecule may comprise four to eight peptide linkers if the multifunctional molecule comprises four cytokines, and at least four "masking" peptide linkers as defined herein.

[0140] The multifunctional molecule comprises at least one "masking" peptide linker by cytokine, said "masking" peptide linker being 5 to 30 amino acids in length and consists of

[0141] - at least 20, 30, 40, 50, 60, 70, 80 or 90% acidic or amidic amino acids independently selected from the group consisting of E, D and N,

[0142] - up to 5, 10 or 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and

[0143] - at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.

[0144] When the peptide linker comprises a ratio or percentage of the amino acids, it is meant that the number of occurrences of this particular amino acid or these particular amino acids to the total number of amino acids in the peptide linker. For instance, in a peptide linker EEEEDEEEEDEEEEDEEEED (SEQ. ID NO: 15), the percentage of acidic or amidic amino acids is 100% (4 D + 16 E / 20 aa), the percentage of basic amino acids is 0% and there is no remaining amino acids. In an additional example, in a peptide linker EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), the percentage of acidic or amidic amino acids is 80% (16 E / 20 aa), the percentage of basic amino acids is 0% and 100% of the remaining amino acids are A. In a last example, in a peptide linker EEEEKEEEEKEEEEK (SEQ ID NO: 46), the percentage of acidic or amidic amino acids is 80% (12 E / 15 aa), the percentage of basic amino acids is 20% (3 K / 15 aa), and there is no remaining amino acids.

[0145] More particularly, when the linker peptide comprises 5, 10 or 20% basic amino acids selected from the group consisting of H, K and R, it has to comprise at least 15, 30 or 60% of acidic or amidic amino acids; respectively.

[0146] The peptide linker comprises at least 5, 6, 7, 8, 9 or 10 amino acids. Preferably, the peptide linker is from 5 to 30 amino acids in length or 10 to 25 amino acids in length, preferably from 10 to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids in length. Even more preferably, the peptide linker is from 10 to 20 amino acids in length.

[0147] Optionally, the peptide linker is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In a particular aspect, the peptide linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In a very particular aspect, the peptide linker is 15 amino acids in length.

[0148] In a preferred aspect, the "masking" peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50, 60, 70, 80 or 90% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; and a sequence having 5 to 30 consecutive amino acids N.

[0149] For instance, in a peptide linker EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), the percentage of polar, amidic and acidic amino acids is 100% (4 D + 16 E / 20 aa) and 0% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T. In an additional example, in a peptide linker EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), the percentage of polar, amidic and acidic amino acids is 80% (16 E / 20 aa) and 20% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T (4 A / 20 aa). In a last example, in a peptide linker EEEETEEEETEEEETEEEET (SEQ ID NO: 21), the percentage of polar, amidic and acidic amino acids is 100% (16 E + 4 T / 20 aa) and 20% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T (4 T / 20 aa). In a particular aspect, a sequence has at least 50, 60, 70, 80 or 90% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and 10, 20, 30, 40 or 50% of remaining amino acids are independently selected from the group consisting of G, P, A, V, S, and T.

[0150] In a particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of:

[0151] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids selected independently from the group consisting of E, D and N; or

[0152] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being E; or

[0153] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being D; or

[0154] - 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being N.

[0155] The "masking" peptide linker(s) envisioned herein essentially consist(s) of or consist(s) of the same amino acid residue selected in the group consisting of Asparagine (N), Glutamate (E) and Aspartic acid (D).

[0156] Preferably, the same amino acid is selected from the group consisting of the group consisting of N, E and D. More preferably, the same amino acid is selected from the group consisting of E, D and N. Even more preferably, the same amino acid is E.

[0157] In the "masking" peptide linker, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% or 100% of the amino acids of the linker can be the same amino acid. In a particular aspect, the multifunctional molecule comprises at least one "masking" peptide linker having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% of the amino acid E, D and N, preferably E.

[0158] In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ. ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46).

[0159] In a preferred aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).

[0160] In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).

[0161] In a even more particular aspect, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).

[0162] In a particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5) and EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), optionally with 1, 2 or 3 amino acid modifications, in particular an amino acid substitution with an amino acid selected from the group consisting of D, N, Q, T, S, G, P, A, V, K, H and R, preferably D, N, Q, T, A and K, even more preferably A, T, D and N.

[0163] Even more preferably, the multifunctional molecule comprises 1, 2, 3, 4, 5, 6, 7 or 8 "masking" peptide linker(s) as defined above.

[0164] In some aspects, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),

[0165] NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46).

[0166] In a preferred aspect, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).

[0167] In a very particular aspect, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), 1

[0168] EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).

[0169] In an even more particular aspect, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, "masking" peptide linker(s) selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).

[0170] In a preferred aspect, a "masking" peptide linker as defined herein is linked at the N-terminal end of the cytokine. Preferably, the peptide linker is covalently linked to the N-terminal end of the cytokine and is covalently linking or connecting the antigen binding domain and the cytokine. Optionally, the multifunctional molecule comprises only one "masking" peptide linker per cytokine.

[0171] Optionally, the multifunctional molecule comprises two peptide linkers per cytokine, a first peptide linker being a "masking" peptide linker as described herein and a second peptide linker, wherein:

[0172] - the second peptide linker has an amino acid sequence as defined herein for a "masking" linker; or

[0173] - the second peptide linker is identical to the first peptide linker; or

[0174] - the second peptide linker is between 10 and 30 amino acids in length and consists of less than 10, 20, 30, 40 or 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.

[0175] For instance, in a peptide linker AAAAHAAAAAAAAAA (SEQ ID NO: 3), the percentage of basic amino acids is 7% (1 H / 15 aa), and the percentage of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T is 93% (14 A / 15 aa). In an additional example, in a peptide linker PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), the percentage of basic amino acids is 0%, and the percentage of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T is 100% (16 P + 4 S / 15 aa).

[0176] In a particular aspect, the second or additional peptide linker consists of less than 10, 20, 30, 40 or 50% of basic amino acids and the remaining amino acids are independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.

[0177] Preferably, the second or additional peptide linker(s) is / are not cleavable or does / di not contain any protease cleavage site. In some aspect, the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I I I TH (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), I I I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I TTH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).

[0178] When the multifunctional molecule comprises more than one peptide linker, the peptide linkers may have the same length or they can have a length slightly different, for instance having a difference in length of at most 10 to 20%, preferably 10% to 15%, in number of amino acids.

[0179] In a preferred aspect, the multifunctional molecule comprises two peptide linkers per cytokine and a first peptide linker being a "masking" peptide linker as defined herein is linked at the N-terminal end of the cytokine and a second peptide linker is linked at the C-terminal end of the cytokine, said second linker being as defined above. In a more preferred, the multifunctional molecule comprises two peptide linkers per cytokine and a first peptide linker is linked at the N-terminal end of the cytokine and a second peptide linker is linked at the C-terminal end of the cytokine, the first and second peptide linkers are being a "masking" peptide linker as defined herein.

[0180] Optionally, the multifunctional molecule comprises linker(s) and a cytokine with one of the following arrangements:

[0181] "masking" linker - cytokine - "masking" linker,

[0182] "masking" linker - cytokine, cytokine - "masking" linker, masking" linker - cytokine - linker, and linker - cytokine - "masking" linker.

[0183] Preferably, the multifunctional molecule comprises linker(s) and a cytokine with one of the following arrangements:

[0184] "masking" linker - cytokine - "masking" linker,

[0185] "masking" linker - cytokine, and masking" linker - cytokine - linker.

[0186] When the cytokine is a dimer, either a homodimer or a heterodimer cytokine, the two monomers of the cytokine are covalently linked by a peptide linker or spacer, optionally a "masking" peptide linker as defined herein. Accordingly, the multifunctional molecule comprises linker(s) and a dimeric cytokine with one of the following arrangements: "masking" linker - 1stmonomer of the cytokine - peptide linker / spacer - 2ndmonomer of the cytokine - "masking" linker,

[0187] "masking" linker - 1stmonomer of the cytokine - peptide linker / spacer - 2ndmonomer of the cytokine - linker,

[0188] "masking" linker - 1stmonomer of the cytokine - peptide linker / spacer - 2ndmonomer of the cytokine,

[0189] "masking" linker - 1stmonomer of the cytokine - "masking" linker - 2ndmonomer of the cytokine - "masking" linker,

[0190] "masking" linker - 1stmonomer of the cytokine - "masking" linker - 2ndmonomer of the cytokine - linker, and

[0191] "masking" linker - 1stmonomer of the cytokine - "masking" linker - 2ndmonomer of the cytokine.

[0192] In some aspects, the multifunctional molecule comprises at least one, preferably 1, 2, 3 or 4, peptide linker(s) and at least one or two peptide linkers are "masking" peptide linkers selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),

[0193] DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),

[0194] NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43), and more preferably EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44), and optionally an additional linker selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), T l I I I I I I I I I I I I H (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I TTH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).

[0195] In a preferred aspect, the multifunctional molecule comprises at least one or two peptide linkers per cytokine selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12),

[0196] EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),

[0197] DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),

[0198] NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),

[0199] DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),

[0200] NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43), and more preferably EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).

[0201] In the present invention, the multifunctional molecule comprises one or more cytokine(s), variant(s) or fragment(s) thereof having an anti-inflammatory effect. Examples of such cytokines are provided here below.

[0202] As described above, in the multifunctional molecule described herein, the peptide linkers of the invention enable to mask and / or demask the cytokine. Preferably, when the cytokine is masked, the cytokine is not able to bind to its cognate receptor and / or does not exhibit its biological activity. Preferably, when the cytokine is unmasked, the cytokine is able to bind to its cognate receptor and / or is able to exhibit its biological activity. The receptors and biological activity of a particular cytokine have been described in the art and are well known to the man skilled in the art. The biological activity of a cytokine typically refers to the effects a cytokine exerts on target cells, tissues, or organs, especially by binding to its receptor and / or triggering intracellular signaling pathways. These activities can include regulating immune responses, inflammation, cell growth, differentiation, survival, or death.

[0203] Cytokines are small protein messengers secreted primarily by immune cells that transmit signals between cells to regulate and orchestrate various immune and inflammatory processes within the body. As used herein, the terms "cytokine having an anti-inflammatory effect" or "anti-inflammatory cytokine" are used interchangeably and refer to a cytokine that is secreted by immune cells inhibit or decrease inflammation. Detecting inflammation can be assessed by a variety of techniques known to the man skilled in the art, such as clinical assessment, biopsy, laboratory tests (e.g., CRP, ESR, WBC), imaging studies (CT or MRI).

[0204] In a specific aspect, the cytokine has an inhibitory effect. More specifically, the cytokine has a inhibitory effect on cells activating the immune response. Alternatively, the cytokine can have a stimulatory effect on cells inhibiting the immune response, such as T reg.

[0205] In a particular aspect, the cytokine having an anti-inflammatory is an immuno-repressing cytokine. The immuno-repressing cytokine is capable of inactivating or repressing an immune cell. The immune cell can be selected in the non-exhaustive list comprising B cells, T cells, in particular CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells and monocytes. In a preferred aspect, the immune cells are T cells, more specifically CD8+ T cells, effector T cells or exhausted T cells. In a particular preferred aspect, the immune cells are effector memory stem like T cells.

[0206] Optionally, the cytokine may have a dual effect. By dual effect, it is intended to refer to a cytokine which can have both a pro-inflammatory effect and an anti-inflammatory effect, depending on the biological context.

[0207] Indeed, some cytokines may exhibit either a pro-inflammatory effect or an anti-inflammatory effect depending on several factors. For example, IL-2 may have a pro-inflammatory effect when directed to Th through signal amplification. On the other hand, when addressed to Treg, it will activate those cells and switch them toward an anti-inflammatory pathway, thus triggering the resolution of the inflammation or the resolution of the auto-immune condition. Other examples exist: IL-10, which may activate B cells and T DC8+ cells which exert a pro-inflammatory effect, or activate myeloid cells and triggering an anti-inflammatory response. It is well known for the person skilled in the art, based on the literature, which combination of cytokine and antigen binding domain may be used to trigger one or the other pathway.

[0208] Optionally, the cytokine may be mutated or altered so that the biological activity of the cytokine is altered, e.g., the biological activity is increased or decreased. In a particular aspect, the cytokine is modified to decrease its biological activity, e.g., its affinity for its receptor.

[0209] Optionally, the cytokine consists in a fragment thereof retaining the biological activity of the cytokine. In one aspect, the cytokine, variant or fragment thereof has a size of at least 10 kDa, at least, 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa or at least 50kDa. Preferably, the cytokine, variant or fragment thereof has a size comprised between 10 kDa and 50 kDa, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 kDa and 20 kDa, between 20 kDa and 50 kDa, between 20 kDa and 40 kDa or between 20 kDa and 30 kDa.

[0210] Preferably, the cytokine is from human or derived from human. In some aspects, the cytokine is an interleukin. In one aspect, the cytokine is a monomer. In an alternative aspect, the cytokine comprises a plurality of subunits. Preferably, the cytokine is a dimer, either a homodimer and a heterodimer, and comprises two subunits. Preferably, if the cytokine has a plurality of subunits, said subunits are linked together by a peptide linker or spacer as described herein. In a preferred aspect, the peptide linker or spacer is linked to the N-terminal end of a first subunit of the cytokine, and to the C-terminal end of a second subunit of the cytokine. In an alternative aspect, the peptide linker or spacer is linked to the C-terminal end of a first subunit of the cytokine, and to the N-terminal end of a second subunit of the cytokine.

[0211] In a particular aspect, the anti-inflammatory cytokine, variant or fragment thereof is a cytokine selected from the group consisting of TGFP, IL-2, IL-4, IL-10, IL-11, IL-12, IL-13, IL-14, IL-19, IL-22, IL-24, IL-25, IL-27, IL-35, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0212] Optionally, the anti-inflammatory cytokine or fragment thereof is a cytokine selected from the group consisting of TGFP, IL-13, IL-19, IL-35, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0213] Optionally, the anti-inflammatory cytokine or fragment thereof is a cytokine selected from the group consisting of IL-2, IL-4, IL-10, IL-11, IL-12, IL-14, IL-22, IL-24, IL-25 and IL-27 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0214] In a very particular aspect, the cytokine is selected from the group consisting of IL-2, IL-10, IL-35, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0215] In particular, the cytokine can be selected in the list of Table D below, or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0216] Table D: List of immunorepressing or anti-inflammatory cytokines

[0217] Optionally, the cytokine is a monomeric cytokine and is selected from the group consisting of TGFP, IL- 2, IL-4, IL-10, IL-11, IL-13, IL-14, IL-19, IL-22, IL-24, IL-25, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof. In a particular aspect, the cytokine is a monomeric cytokine and is selected from the group consisting of IL-2, IL-10, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0218] Optionally, the cytokine is a monomeric cytokine and is selected from the group consisting of IL-12, IL- 27, IL-35, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof. In a particular aspect, the cytokine is a monomeric cytokine and is IL-12, IL-35 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0219] In a very specific aspect, the immuno-stimulating cytokine is IL-2 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0220] In another very specific aspect, the immuno-stimulating cytokine is IL-10 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0221] In another very specific aspect, the immuno-repressing cytokine is IL-35 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0222] In another very specific aspect, the immuno-repressing cytokine is IL-37 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0223] In another very specific aspect, the immuno-repressing cytokine is IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0224] Preferably, the cytokine variant or fragment described herein retains substantially equivalent biological property and / or function in comparison to a wild-type cytokine For instance, it retains comparable biological property / function as the full-length or wild-type protein, respectively. Preferably, the cytokine variant or fragment maintains biological activity of at least 10 %, 20%, 30%, 40%, 50%, 60% in comparison with the wild type cytokine. Preferably, the biological activity is the bind of the cytokine to its receptor and / or inhibition of said receptor. Preferably, the cytokine variant is a function conservative variant.

[0225] In a particular aspect, the cytokine has a narrow therapeutic index. Therapeutic index (Tl) is the range of doses at which a medication is effective without unacceptable adverse events. Narrow therapeutic index (NTI) cytokines are defined as those cytokines where small differences in dose or blood concentration may lead to dose and blood concentration dependent, serious therapeutic failures or adverse drug reactions. Serious events are those which are persistent, irreversible, slowly reversible, or life-threatening, possibly resulting in hospitalization, disability, or even death. Particularly preferred cytokines beneficiating from the masking effect described herein are those having a narrow therapeutic index. A drug product typically has a narrow therapeutic index when:

[0226] (a) there is less than a 5-fold, 4-fold, 3-fold or 2-fold difference in median lethal dose (LD50) and median effective dose values (ED50) and / or

[0227] (b) there is less than a 5-fold, 4-fold, 3-fold or 2-fold difference in the minimum toxic concentrations (MTC) and minimum effective concentrations (MEC) in the blood and / or

[0228] (c) safe and effective use of the drug requires careful titration and patient monitoring.

[0229] Preferably, there is (a) less than a 2-fold difference in median lethal dose (LD50) and median effective dose values (ED50) and / or (b) less than a 2-fold difference in the minimum toxic concentrations (MTC) and minimum effective concentrations (MEC) in the blood.

[0230] In particular, the cytokines with a NTI could be selected in the non-exhaustive list comprising TGFP, IL- 2, IL-4, IL-10, IL-11, IL-12, IL-13, IL-14, IL-19, IL-22, IL-24, IL-25, IL-27, IL-35, IL-37 and IL-38.

[0231] IL-2 and IL-2 variants

[0232] In a very specific aspect, the immuno-repressing cytokine is lnterleukin-2 (IL-2), preferably a human IL- 2, for example as disclosed under the UniProt accession number P60568 or a mutant or variant thereof. The IL-2 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 50 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 50.

[0233] IL-2 can be mutated in various ways to reduce its toxicity and / or increase its efficacy. Hu et al. (Blood 101, 4853-4861 (2003), US Pat. Publ. No. 2003 / 0124678) have substituted the arginine residue in position 38 of IL-2 by tryptophan to eliminate IL-2's vasopermeability activity. Shanafelt et al. (Nature Biotechnol 18, 1 197-1202 (2000)) have mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); US Pat. No. 5,229, 109) have introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of proinflammatory cytokines from NK cells. Gillies et al. (US Pat. Publ. No. 2007 / 0036752) have substituted three residues of IL-2 (Asp20Thr, Asn88Arg, and Glnl26Asp) that contribute to affinity for the intermediate-affinity IL- 2 receptor to reduce VLS. Gillies et al. (WO 2008 / 0034473) have also mutated the interface of IL-2 with CD25 by amino acid substitution Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells for enhancing efficacy. In one aspect, the cytokine is an IL-2 mutant for example as described in WO 2012 / 107417 or WO 2018 / 184964.

[0234] Optionally, the IL-2 variant may comprise one or several substitutions at positions of human IL-2 (without the signal peptide; SEQ ID NO: 50) selected from the group consisting of Qll, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, 192, T123, Q126, SI 27 , 1129, and S130, the numbering being preferably the one of SEQ ID NO: 50. Optionally, the IL-2 variant may comprise the substitution relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) F42A or F42K. Optionally, the IL-2 variant may further comprise one or several substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) selected from the group consisting of: R38A, R38D, R38E, E62Q, E68A, E68Q, E68K and E68R, and / or

[0235] H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, SI 27 A, S127E, S127K, and S127Q, and / or

[0236] C125A, the numbering being the one of SEQ ID NO: 50.

[0237] Optionally, the IL-2 variant may comprise one of the following substitutions combination relative to human IL-2 (without the signal peptide; SEQ ID NO: 50): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and V91E; R38E, F42A, and D84H; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and V91E; R38D, F42A, and D84H; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and V91E; R38A, F42K, and D84H; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and V91E; F42A, E62Q, and D84H; H16D, F42A, and E62Q; H16E, F42A, and E62Q; F42A, E62Q, and Q126S; R38E, F42A, and C125A; R38D, F42A , and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A , C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A; F42A, C125A and Q126S; F42A, Y45A and L72G; and T3A, F42A, Y45A, L72G and C125A, the numbering being preferably the one of SEQ ID NO: 50.

[0238] Optionally, the IL-2 variant may comprise one of the following substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50), in particular at least one of the substitutions selected in the group comprising K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K; or a combination thereof, preferably the three substitutions F42A, Y45A and L72G, the numbering being preferably the one of SEQ ID NO: 50.

[0239] Mutants of human IL-2 (hlL-2) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 3, 35, 38, 42, 43, 45 or 72 or combinations thereof, corresponding to residues position of human IL-2 (without the signal peptide; SEQ ID NO: 50). Preferably, the mutant IL-2 is a human IL-2 molecule comprising the amino acid substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) T3A, F42A, Y45A, L72G and / or C125A, preferably F42A, Y45A and L72G, more preferably T3A, F42A, Y45A, L72G and C125A, for example as disclosed in WO 2018 / 184964. Even more preferably, the immuno-repressing cytokine is an IL-2 mutant having the substitutions relative to human IL-2 (without the signal peptide; SEQ ID NO: 50) F42A, Y45A and L72G, preferably T3A, F42A, Y45A, L72G and C125A, the numbering being the one of SEQ ID NO: 50.

[0240] IL-10 and IL-10 variants

[0241] In a very specific aspect, the immuno-repressing cytokine is Interleukin-10 (IL-10), preferably a human IL-10, or a fragment or variant thereof.

[0242] As used herein, the terms "IL-10", "interleukin 10" or "Cytokine synthesis inhibitory factor (CSIF)" are used interchangeably. For example, IL-10 is disclosed under the UniProt accession number P22301. The mature polypeptide sequence of the IL-10 protein which is 160 amino acids long is disclosed in SEQ ID NO: 51.

[0243] IL-10 variants are known in the field. For instance, IL-10 variants are disclosed in WQ2020108426, US2023192796, WO23102493, WO22149881, WQ2021006605, WQ04056850, WQ23060165, the disclosure of which being incorporated herein by reference.

[0244] The IL-10 variant preferably has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the wildtype cytokine of SEQ ID NO: 51 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof with respect to the sequence of SEQ ID NO: 51. Preferably, the IL-10 fragment, variant or mutant is capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or IL-10 receptor binding activity of a wild-type IL-10.

[0245] Non-exhaustively, IL-10 variants may include one or more amino acid substitution selected from the group consisting of P2A, N10Q, N21A, M22A, M39T, I87A, N92Q, D25A, D25K, E96A, E96K, R104A, the numbering being preferably the one of SEQ ID NO: 51. The IL-10 variants may comprise an amino acid substitution at position 99; in particular, may comprise an amino acid substitution at position 99, where this substitution is amino acid N. The IL-10 variants may comprise at least two amino acid substitutions at positions 18, 92 and 99; in addition, they may comprise one or more further substitutions at positions 55, 69, 97, 110, 111 and / or 148, the numbering preferably being the one of SEQ ID NO: 51.

[0246] In some embodiments, IL-10 variants may include one of the following sets of mutations: D25A and E96A; N21A and R104A; N21A and D25A; N21A, D25A and E96A; or N21A, M22A and D25A the numbering preferably being the one of SEQ ID NO: 51.

[0247] IL-10 variants may include one of the following sets of mutations: N18Y, N92Q, T100D, and R104W; N18Y, N21H, N92Q, E96D, T100V, and R104W; N18Y, N21H, N92Q, E96H, T100V, and R104W; N18Y, D25A, N92Q, T100D, and R104W; N18Y, D25K, N92Q, T100D, and R104W; or N18Y, D25A, N92Q, E96A, T100D, R104W, the numbering being preferably the one of SEQ ID NO: 51.

[0248] IL-10 variants may comprise one or more amino acid substitutions at a position corresponding to residues H14, N18, N21, M22, D25, R32, S93, E96, and T100, the numbering being preferably the one of SEQ ID NO: 51. In particular, IL-10 variants may comprise one or more amino substitutions at a position corresponding to amino acid residues selected from the group consisting of H14, N18, N21, M22, D25, R32, S93, E96, and T100, wherein: the amino acid substitution at position H14 is selected from the group consisting of H14C, H14F, H14P, H14W and H14G; the amino acid substitution at position N18 is selected from the group consisting of N18R and N18K; the amino acid substitution at position N21 is selected from the group consisting of N21C, N21D and N21E; the amino acid substitution at position M22 is selected from the group consisting of M22D, M22S, M22T, and M22W; the amino acid substitution at position D25 is selected from the group consisting of D25P and D25Q; the amino acid substitution at position R32 is selected from the group consisting of R32N, R32Q, R32G, R32C, R32P, R32F, and R32Y; the amino acid substitution at position S93 is S93G; the amino acid substitution at position E96 is selected from the group consisting of E96C, E96F, E96Y, and E96W; and the amino acid substitution at position T100 is T100C, the numbering being preferably the one of SEQ ID NO: 51.

[0249] IL-10 variants may comprise one or more amino substitutions selected from the group consisting of: H14A, HMD, H14E, H14I, H14K, H14L, HMM, H14N, H14Q, H14R, H14S, H14T, H14Y, and H14V; N18Y, N18F, N18A, N18D, N18E, N18L, N18V, N18S, N18T, N18I, N18V, N18M, and N18H; N21A, N21R, N21Q, N21H, N21K, N21S, N21V, N21I, N21L, N21M, and N21T; M22A, M22V, M22I, M22L, M22N, and M22Q; R24E, R24D, R24N, R24Q, R24A, R24S, and R24T; D25A, D25N, D25H, D25I, D25K, D25L, and D25V; D28A, D28E, D28L, D28V, D28S, D28T, D28I, D28V, D28M, D28H, D28K, and D28R; R32A, R32D, R32E, R32L, R32V, R32S, R32T, R32I, R32V, R32M, and R32H; E74A, E74D, E74L, E74V, E74S, E74T, E74I, E74V, E74M, E74H, E74K, and E74R; H90A, H90D, H90E, H90I, H90K, H90L, H90M, H90N, H90Q, H90R, H90S, H90T, H90Y, and H90V; N92D, N92Q, N92E, N92H, N92K, N92S, N92V, N92I, N92L, N92M, N92T, and N92A; S93E, S93A, S93R, S93N, S93D, S93Q, S93E, S93I, S93L, S93K, S93M, and S93V; E96A, E96N, E96D, E96Q, E96H, E96K, and E96S; T100D, T100V, T100E, T100A, T100R, T100N, T100Q, T100E, T100I, T100L, T100K, T100M, and T100S; or R104A, R104W, R104Y, R104F, R104H, R104D, R104E, R104N, R104Q, R104S, R104T, R104I, R104L, R104V, and R104M, the numbering being preferably the one of SEQ ID NO: 51.

[0250] IL-10 variants may comprise an amino acid substitution on amino acids in position 104, position 107, or a combination thereof. In particular, the amino acids substitutions can be R104Q, any one of R107A, R107E, R107Q and R107D, or a combination thereof, the numbering being preferably the one of SEQ ID NO: 51.

[0251] IL-35 and IL-35 variants

[0252] In a very specific aspect, the immuno-repressing cytokine is Interleukin-35 (IL-35), preferably a human IL-35, or a fragment or variant thereof.

[0253] Interleukin-35 (IL-35) is a member of the IL-12 family of heterodimeric cytokines and is composed of Ebi3, a b chain subunit encoded by the Epstein-Barr virus (EBV)-induced gene 3 (also known as I L27b), and the I L12 p35 a subunit encoded by I L-12a. IL-35 is produced by regulatory T cells and is involved in the immunosuppressive activities of Tregs.

[0254] As used herein, the terms "p35", "IL12-A", "Cytotoxic lymphocyte maturation factor 35 kDa subunit (CLMF p35)", "IL-12 subunit p35' and "NK cell stimulatory factor chain 1 (NKSF1)" are used interchangeably and refer to the p35 subunit of IL-35. Examples of p35 sequences are provided in Uniprot P29459 and in GenBank Accession NP_000873.2, NM_000882.3, NM_008351.3, NM_001159424.3, NM_001397992.1, NM_001354582.2, NM_001354583.2 and NM_000882.4.

[0255] Preferably, the p35 subunit comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 53.

[0256] As used herein, the terms "EBi3", "EBI3", "IL-27 subunit beta", "IL-27B", and "Epstein-Barr virus- induced gene 3 protein (EBV-induced gene 3 protein)" are used interchangeably and refer to the Ebi3 subunit of IL-35. Examples of Ebi3 sequences are provided in Uniprot Q14213, ENSMUST00000003274.8, and in GenBank Accession NP_005746.2, NM_005755.2, NM_005755.3 and NM_015766.2. Preferably, the Ebi3 subunit comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 52.

[0257] In one aspect, the IL-35 is a native or wild-type IL-35 protein.

[0258] In some embodiments, the IL-35 comprises a full-length Ebi3 and a full-length p35 subunit. Alternatively, the IL-35 comprises a functional fragment of Ebi3 and / or p35 subunit(s) that is / are capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or some or full receptor binding activity of the full-length IL-35 molecule.

[0259] In some embodiments, the IL-35 is an IL-35 variant or mutant. Such variant typically comprises mutations such as deletion(s), addition(s) and / or substitution(s) in the Ebi3 and / or p35 subunit(s), in particular in the amino acid sequence as set forth in SEQ ID NO: 52 and / or SEQ ID NO: 53, respectively. In some aspects, the p35 subunit is a variant or mutant of SEQ ID NO 53 and has at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Particularly, the p35 subunit is a variant or mutant of SEQ ID NO 53 comprising 1, 2, 3, 4 or 5 mutations such as substitution(s), addition(s) and / or deletion(s), preferably substitution(s).

[0260] Alternatively or additionally, the Ebi3 subunit is a variant or mutant of SEQ ID NO: 52 and has at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Particularly, the Ebi3 subunit is a variant or mutant of SEQ ID NO: 52 comprising 1, 2, 3, 4 or 5 mutations such as substitution(s), addition(s) and / or deletion(s), preferably substitution(s).

[0261] In some aspects, the IL-35 is an IL-35 mutant comprising EBI3 subunit having one or more amino acid substitutions selected from the group consisting of K22, R73, R194, R196, C319, C496, the numbering being preferably the one of SEQ ID NO: 52, or combinations thereof. In some aspects, the IL-35 molecule is as described in WQ2023250459, the disclosure of which being incorporated by reference. Preferably, the IL-35 variant or mutant is capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or IL-35 receptor binding activity of a wild-type IL-35. In some embodiments, the IL-35 variant is a modified IL-35, such as glycosylated IL- 35.

[0262] An IL-35 variant, mutant or molecule comprising a functional fragment of Ebi3 and / or p35 subunit(s), is considered capable of producing some producing some or full biological activity and / or IL-35 receptor binding activity of a wild-type IL-35 when it reduces the expression of one or more antiinflammatory cytokine selected from the group comprising IL12p40, IL2 and IL6, and / or reduces the expression of one or more transaminase enzyme selected from the group comprising Aspartate aminotransferase (ASAT) and Alanine aminotransferase (ALAT) by 10%, preferably 20%, more preferably 30%, more preferably 40%, more preferably 60%, more preferably 70%, even more preferably 75%, in particular in comparison to the expression of said anti-inflammatory cytokine or transaminase enzyme without IL35. Preferably, an IL-35 variant, mutant or molecule comprising a functional fragment of Ebi3 and / or p35 subunit(s), is considered capable of producing some producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or IL-35 receptor binding activity of a wild-type IL-35 when it reduces the expression of one or more anti-inflammatory cytokine selected from the group comprising IL12p40, IL2 and IL6, and / or reduces the expression of one or more transaminase enzyme selected from the group comprising Aspartate aminotransferase (ASAT) and Alanine aminotransferase (ALAT) by 10%, preferably 20%, more preferably 30%, more preferably 40%, more preferably 60%, more preferably 70%, even more preferably 75%, in particular in comparison to a wildtype IL-35.

[0263] In some embodiments, the IL-35 can contain a portion of a native protein from a human and a portion of a native protein from a mouse (i.e., a "chimeric" protein).

[0264] Preferably, the two subunits of IL-35 are covalently linked by a peptide linker or peptide spacer. Preferably, they are covalently linked by a "masking" peptide linker as defined herein.

[0265] IL-37 and IL-37 variants

[0266] In a very specific aspect, the immuno-repressing cytokine is Interleukin-37 (IL-37), preferably a human IL-37, or a fragment or variant thereof.

[0267] As used herein, the terms "IL-37", "interleukin 37", "IL-1 family member 7 ", "IL-1F7", "IL-1H4", IL- 1RP1" are used interchangeably and refer to the seventh member of the IL-1 family discovered by computational cloning in 2000, which was renamed in 2010 (Dinarello C, et al. Nat Immunol. 2010 Nov; 11(11):973). IL-37 is also known as interleukin-37 (FILI zeta; IL-1 zeta; IL-1 F7b (IL-1 H4, IL-1 H, IL-1 RP1); IL-1X protein; IL1 F7 (canonical product IL-1 F7b); interleukin 1 family member 7; interleukin 1 , zeta; interleukin- 1 homolog 4; interleukin- 1 superfamily z; interleukin- 1 -related protein and interleukin-23). The expression of IL-37 is induced by several toll-like receptor (TLR) ligands and pro- inflammatory cytokines such as IL-1 beta, TNF-alpha and IFN-gamma.

[0268] The structure of IL-37 typically consists of 12 / ? tubular lines. The IL-37 gene particularly encodes 6 exons that undergo alternative splicing: five different splice variants of IL-37 have been identified and termed IL-37a, I L-37b, I L-37c, I L-37d, and IL-37e (Boraschi D et al. Eur Cytokine Netw. 2011 Sep; 22(3): 127-47; Tete S, et al. Int J Immunopathol Pharmacol. 2012 Jan-Mar; 25(l):31-8)., of which I L-37b is the largest (218 amino acids including the propeptide from 1-45) and best characterized isoform yet.

[0269] The term "IL-37", "IL-37 molecule", "IL-37 polypeptide" or "IL-37 protein" as used herein, broadly refers to any native IL-37 from any mammalian source, including primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses "full-length", unprocessed IL- 37 as well as any forms of IL-37 that result from processing in the cell. For example, both full-length IL- 37 and the mature form, wherein the IL-37 is converted from an inactive precursor state into an active state by cleavage of caspase-1 during expression, are encompassed by the present invention. The term also encompasses naturally occurring variants of IL-37, e.g., splice variants or allelic variants, and non- naturally occurring variant or mutants, such as IL-37 truncation or fusion protein.

[0270] In some aspects, the IL-37 is a native or wild-type IL-37 protein.

[0271] Reference IL-37 constructs and / or polypeptides as described herein and known in the art may include but are not limited to those described in W016201503, WO18175403, WO22056897 and WO21123173, W00140247, which are incorporated herein by reference in their entirety.

[0272] In some aspects, the amino acid sequence of IL-37 is as described in GenBank accession number NP_055254.2, NP_775294.1, NP_775295.1, NP_775296.1 or NP_775297.1. In some aspects, the sequence of IL-37 is as described in GenBank accession number NP_055254 46 and consists of a 218 amino acid protein, which is part of the front end of the signal peptide after removal. Of course, the present invention IL-37 may also refer to protein accession number NP_055254, as those skilled in the art know that its front end behind the signal peptide (46-218 amino acids) does not affect the peptide functions. In some aspects, the amino acid sequence of IL-37 is as described in Uniprot accession number Q9NZH6.

[0273] In some aspects, the IL-37 is a native or wild-type IL-37, preferably a human wild-type IL-37. Preferably, the IL-37 has an amino acid sequence comprising or consisting of SEQ ID NO: 54 or having at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0274] Preferably, the IL-37 is selected from the group consisting of IL-37 isoform a, b, c, d and e, preferably a, b and d, even more preferably is IL-37a or I L-37b.

[0275] In some aspects, the IL-37 molecule is the a isoform, i.e., IL-37a or a variant thereof i) having at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto or ii) having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5 mutation(s) selected from the group consisting of addition, deletion and substitution, preferably substitution.

[0276] In some aspects, the IL-37 molecule is the b isoform, i.e., IL-37b or a variant thereof i) having at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto or ii) having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5 mutation(s) selected from the group consisting of addition, deletion and substitution, preferably substitution.

[0277] In some aspects, the IL-37 molecule is the c isoform, i.e., IL-37cor a variant thereof i) having at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto or ii) having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5 mutation(s) selected from the group consisting of addition, deletion and substitution, preferably substitution.

[0278] In some aspects, the IL-37 molecule is the e isoform, i.e., IL-37eor a variant thereof i) having at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto or ii) having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5 mutation(s) selected from the group consisting of addition, deletion and substitution, preferably substitution. In some aspects, the IL-37 is an IL-37 natural variant, such as having a mutation selected from the group consisting of G31V, T42A, P50R, N54S, P108L, R152W, W164R, I177T and D218N, and any combination thereof, the position of amino acid being of the sequence described in Uniprot accession number Q9NZH6, preferably in SEQ ID NO: 54.

[0279] In some aspect, the IL-37 molecule is an IL-37 variant, and the mutation or modification reduces or prevents the polypeptide from forming a dimerization interface. Such modification is capable of preventing dimerization of IL-37 monomers. In general, mutations or modifications are located in the region of the peptide having the same amino acid sequence as the amino acid sequence forming the dimerization interface of the IL-37 monomer. The mutation or modification may be located in the beta 3 or beta 4 ring of the dimerization interface forming the IL-37 monomer. Preferably, the mutation or modification occurs on the residue of T42, K83, N84, Y85, 186, R87 and / or P88, or the residue equivalent to its location, the numbering being preferably as described in SEQ ID NO: 54. Preferably, the mutation is a replacement with a non-conservative amino acid residue, and more preferably, the mutation is a replacement with alanine or an amino acid with an opposite charge. More preferably, the mutation is any one or more of V71A, D73A, D7K, K83E, K83A, Y85A V80A and I78A, the numbering being preferably as described in SEQ ID NO: 54.

[0280] Preferably, the IL-37 variant or mutant is capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or some or full IL-37 receptor binding activity. In some embodiments, the IL-37 variant is a modified IL-37, such as a glycosylated IL-37.

[0281] Preferably, the IL-37 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the anti-inflammatory capacity of a wild-type IL-37. Additionally or alternatively, the IL-37 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the binding capacity to the receptor IL-R8 and / or IL_18Ralpha, in comparison to a wild-type IL-37. Additionally or alternatively, the IL-37 variant or mutant is still able to enhance STAT3 and phosphatase tensin homolog (PTEN) activity.

[0282] In some aspects, the IL-37 can contain a portion of a native protein from a human and a portion of a native protein from a mouse (i.e., a "chimeric" protein).

[0283] Optionally, the two subunits of IL-37 are covalently linked by a peptide linker or peptide spacer. Preferably, they are covalently linked by a "masking" peptide linker as defined herein.

[0284] IL-38 and IL-38 variants

[0285] In a very specific aspect, the immuno-repressing cytokine is Interleukin-38 (IL-38), preferably a human IL-38, or a fragment or variant thereof.

[0286] The terms "IL-38", "interleukin-38", "IL-1 family member 10", "IL1F10", "lnterleukin-1 HY2 (IL-1HY2)" and "lnterleukin-1 theta (IL-1 theta)" are used interchangeably and refer to a member of the interleukin 1 (IL-1) family. IL-38 shares 41% homology with IL-IRa and 43% with IL-36Ra and is known to be an IL-1 receptor antagonist. Structurally, IL-38 is composed of 12 p-strands connected by 11 loops, organized into a p-trefoil configuration.

[0287] The term "IL-38", "IL-38 molecule", "IL-38 polypeptide" or "IL-38 protein" as used herein, broadly refers to any native IL-38 from any mammalian source, including primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses "full-length" IL-38 as well as any forms of IL-38 that result from processing in the cell. The term also encompasses naturally occurring variants of IL-38, e.g., splice variants or allelic variants, and non-naturally occurring variant or mutants, such as IL-38 truncation, dimer or fusion protein.

[0288] In some aspects, IL-38 is a native or wild-type IL-38 protein. Preferably, human IL38 is a precursor protein of 152 amino acids with a predicted weight of 16.9 kDa.

[0289] IL-38 contemplated in this invention are particularly disclosed in W02023102605, WO2019184438, W02016012312, WO2018206496, WO22006588, the content of which is incorporated by reference.

[0290] In some aspects, the amino acid sequence of IL-38 is as described in GenBank accession number NP_115945.4, NP_775184.1, and Uniprot accession number Q8WWZ1, in particular Q8WWZ1-1 and Q8WWZ1-2 or in Uniprot accession number Q8R459.

[0291] In some aspects, the IL-38 molecule is a native or wild-type IL-38, preferably a human wild-type IL-38. Preferably, the IL-38 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 55 or a sequence having at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0292] In some aspects, the IL-38 is a precursor of IL-38. In some aspects, the IL-38 is a truncated IL-38.

[0293] The IL-38 molecule may envisioned herein may be of several forms: a full-length form consisting of 152 amino acids (1st to 152th amino acid), a truncated form consisting of 149 amino acids (3rd to 152th amino acid) or a truncated form consisting of 133 amino acids (20th to 152th amino acid).

[0294] As used herein, the term "truncated IL-38" or "truncated IL-38 protein" refers to an IL-38 polypeptide in which amino acid residues have been removed from the amino-terminus (or N-terminal) area of the full length IL-38 polypeptide.

[0295] In some aspects, the truncated IL-38 protein of the invention has 2 to 50 amino acids, truncated at its N-terminus, as compared with a wild type IL-38 protein (e.g., SEQ ID NO: 55). Preferably said truncated IL-38 protein has 3, 5, 7, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids truncated at its N- terminal as compared to the protein shown in SEQ ID NO: 55.

[0296] Preferably, the IL-38 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 55 or i) has at least / more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto or ii) has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations selected from the group consisting of addition, deletion and substitution, preferably substitution. Preferably, the IL-38 variant or mutant is capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or some or full IL-38 receptor binding activity.

[0297] Preferably, the IL-38 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the anti-inflammatory capacity of the wild-type IL-38.

[0298] Preferably, the IL-38 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% of the capacity to reduces the induction of IL-ip, TNF, IL-17, IL-6, IL-23, and MCP-1, in comparison to a wild-type IL-38.

[0299] Additionally or alternatively, the IL-38 variant or mutant retains at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99% capacity to bind the receptor IL1RAPL1 and / or IL-36R.

[0300] In some embodiments, the IL-38 variant is a modified IL-38, such as glycosylated IL-38.

[0301] In some aspects, the IL-38 molecule is a monomer of IL-38. Alternatively, the IL-38 molecule is a homodimer of IL-38.

[0302] In some aspects, the IL-38 molecule is an IL-38 that is not cleavable by proteases, in particular endogenous proteases.

[0303] Particularly, the present invention provides a polypeptide comprising an amino acid sequence of an IL- 38, the amino acid sequence having a mutation or modification that increases the stability of the monomer and thereby typically reduces the likelihood that the polypeptide will form a homodimer.

[0304] Preferably, the mutation or modification reduces or prevents the polypeptide from forming a dimerization interface that enables dimerization of IL-38 monomers. Typically, the mutation or modification is located in a region of the polypeptide that has the same amino acid sequence as the amino acid sequence that forms the dimerization interface of an IL-38 monomer.

[0305] Preferably, mutation or modification may be of an amino acid residue located in the domain-swap hinge region of IL-38. Particularly, mutation or modification may be of any residue which disrupts the alpha-helical structure that forms in the IL-38 polypeptide, enabling formation of the IL-38 homodimer and which constitutes the domain-swap hinge region.

[0306] For example, the substitution or modification is preferably one which promotes formation of an IL-38 monomer over an IL-38 homodimer (such that the equilibrium between formation of dimer and monomer is driven towards formation of the monomer). More preferably, the substitution or modification is one which allows for stabilization of the IL-38 monomer, such that little or no homodimer is formed. Preferably the substitution is to an amino acid that prevents or reduces the capacity for formation of alpha-helical structure in the IL-38 monomer (for example, in the region corresponding to residues 43 to 58 of SEQ ID NO: 55).

[0307] In another aspect, IL-38 has a reduced capacity to form a dimer compared to a polypeptide having the sequence of SEQ ID NO: 55, wherein the polypeptide has a modification in the region corresponding to residues 43 to 58 of SEQ ID NO: 55. Modification includes, deleting the loop, shortening the loop, lengthening the loop, mutating one or more residues of the loop and / or chemically modifying one or more residues of the loop.

[0308] More preferably, the substitution or modification is of a residue at a position, or at a position equivalent to, C43, 144, L45, P46, N47, R48, G49, L50, A51, R52, T53, K54, V55, P56, 157 or F58 of SEQ ID NO: 55 and, wherein the substitution or modification allows for stabilization of the IL-38 monomeric form.

[0309] In some embodiments, the IL-38 molecule comprises a mutation selected from the group consisting of I44T, A51D, T53N, V55Q, or V55T, and any combination thereof, the amino acid number corresponding to the amino acid position of SEQ ID NO: 55.

[0310] In some particular embodiments, the IL-38 molecule is a truncated IL-38 molecule and comprising a mutation selected from the group consisting of I44T, A51D, T53N, V55Q, or V55T, and any combination thereof, the amino acid number corresponding to the amino acid position of SEQ ID NO: 55.

[0311] In some embodiments, the IL-38 can contain a portion of a native protein from a human and a portion of a native protein from a mouse (i.e., a "chimeric" protein).

[0312] Optionally, the two subunits of IL-37 are covalently linked by a peptide linker or peptide spacer. Preferably, they are covalently linked by a "masking" peptide linker as defined herein.

[0313] In some specific aspects, in the multifunctional molecule of the invention, the cytokine is IL-2 or IL-10 or a variant thereof, typically such as described here above.

[0314] In some aspects, the multifunctional molecule of the invention can comprise 1, 2, 3 or 4 cytokine molecules, that can be the same or different. When the multifunctional molecule comprises more than one cytokine, the cytokines are preferably attached in series to one another and connected by a peptide linker or spacer according to the invention.

[0315] Preferably, the multifunctional molecule of the invention comprises a single cytokine molecule.

[0316] Antigen binding domain

[0317] The antigen binding domain or targeting moiety of the multifunctional molecule is an antibody, an antigen binding fragment thereof or a derivative thereof.

[0318] In a first aspect, the antigen binding domain comprised in the multifunctional molecule of the invention specifically binds to a target expressed on immune cells surface, particularly targets that are only or specifically expressed on immune cells.

[0319] In another particular aspect, the antigen binding domain comprised in the multifunctional molecule of the invention is directed towards a target not expressed on tumoral cells.

[0320] In particular, the antigen binding domain comprised in the multifunctional molecule does not bind or have affinity to the cytokine. As used herein, an "antigen-binding fragment" or "antigen-binding domain" of an antibody means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for a particular antigen, possibly in its native form; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragments have a similar binding affinity as the corresponding 4- chain antibodies. However, antigen-binding fragment that have a reduced antigen-binding affinity with respect to corresponding 4-chain antibodies are also encompassed within the invention. The antigenbinding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be designated as "functional fragments" of antibodies. Antigen-binding fragments of antibodies are fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof. Antigen binding domain fragment typically includes Fab domain, Fab', (Fab')2. Antigen binding domain derivatives typically includes single-chain variable fragment (scFV), scFab, diabody and CrossMAb.

[0321] With regard to the "binding" capacity of the antigen binding domain, the terms "bind" or "binding" refer to antibodies including antigen binding fragments and derivatives thereof that recognize and contact another peptide, polypeptide, protein or molecule. The terms "specific binding", "specifically binds to," "specific for," "selectively binds" and "selective for" a particular target mean that the antigen binding domain recognizes and binds a specific target, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically (or preferentially) binds to an antigen is an antibody that binds the antigen for example with greater affinity, avidity, more readily, and / or with greater duration than it binds to other molecules. Preferably, the term "specific binding" means the contact between an antibody and an antigen with a binding affinity equal or lower than 10’7M. In certain aspects, antibodies bind with affinities equal or lower than IO-8M, IO-9M or IO10M.

[0322] As used herein, the term "target" of the antigen binding domain refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted by the antigen binding domain according to the invention and expressed on the external surface of immune cells. With regards to the expression of a target on the surface of immune cells, the term "expressed" refers to a target, such as carbohydrates, lipids, peptides, polypeptides, proteins, antigens or epitopes that are present or presented at the outer surface of an immune cell.

[0323] The antigen binding domain of the multifunctional molecule can be a Fab domain, a Fab', a F(ab')2, a single-chain variable fragment (scFV), a Fv, a diabody, a scFab, a CrossMAb, a single domain antibody (sdAb) or a VHH. Preferably, the antigen-binding domain is a Fab domain, a single-chain variable fragment (scFV) or a VHH. In some aspects, the antigen-binding domain is a scFV. Optionally, the antigen binding domain can be an antibody such as an IgG antibody that may include modifications, in particular in the Fc domain; a Fab, a Fab', a F(ab')2, a Fv, a diabody, a single domain antibody (sdAb), a VHH, a scFab, a CrossMAb or a ScFv, optionally linked to a Fc domain.

[0324] Optionally, the antigen binding domain can be a Fab domain, a Fab', a (Fab')2, a single-chain variable fragment (scFV), a scFab, a diabody, a CrossMAb, or a single domain antibody (sdAb) or a VHH. The antigen-binding domain preferably comprises a heavy chain variable region (VH) and a light chain variable region (VL). Preferably, the antigen-binding domain is a Fab, a single-chain variable fragment (scFV) or a VHH.

[0325] In some aspects, the antigen binding domain is a CrossMAb. As used herein, the term "CrossMAb" refers to antigen binding domains with an inversion of CL and CHI domains, in particular in one binding arm of antibodies. Thus, such binding domain comprises a VH domain linked to a CL domain and a VL domain linked to a CHI domain. Such format reduces the byproduct formation caused by a mismatch of a light chain of a first binding domain that specifically binds to a first antigen with the wrong heavy chain of a second binding domain that specifically binds to a second antigen (when compared to approaches without such CL-CH1 domain exchanges). CrossMAb are for example described in WO 2009 / 080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191, the disclosure of which being incorporated herein by reference.

[0326] In some aspects, the antigen binding domain is a recombinant antibody or an antigen binding fragment thereof. As used herein, the term "recombinant antibody" refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0327] In some aspects, the antigen binding domain is or derived from an humanized antibody.

[0328] As used herein, the term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., chimeric antibodies that contain minimal sequence derived from a non-human antibody). A "humanized form" of an antibody, e.g., a non- human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. Additional framework region modifications may be made within the human framework sequences. Preferably humanized antibody has a T20 humanness score greater than 80%, 85% or 90%. "Humanness" of an antibody can for example be measured using the T20 score analyzer to quantify the humanness of the variable region of antibodies as described in Gao S H, Huang K, Tu H, Adler A S. BMC Biotechnology. 2013: 13:55 or via a web-based tool to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com.

[0329] In some aspects, the antigen binding domain is or derived from an chimeric antibody.

[0330] By "chimeric antibody" is meant an antibody made by combining genetic material from a nonhuman source, preferably such as a mouse, with genetic material from a human being. Such antibody derives from both human and non-human antibodies linked by a chimeric region. Chimeric antibodies generally comprise constant domains from human and variable domains from another mammalian species, reducing the risk of a reaction to foreign antibodies from a non-human animal when they are used in therapeutic treatments.

[0331] In one aspect, the target is specifically expressed by immune cells in a healthy subject or in a subject suffering from a disease, in particular such as an inflammatory disease or disorder or an auto-immune disease or disorder. This means that the target has a higher expression level in immune cells than in other cells or that the ratio of immune cells expressing the target by the total immune cells is higher than the ratio of other cells expressing the target by the total other cells. Preferably the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100.

[0332] "Immune cells" as used herein includes neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells). It preferably refers to T cells, more specifically CD4+ T cells, CD8+ T cells, effector T cells and / or exhausted T cells.

[0333] By "activated immune cells" it is meant immune cells that are involved or have been activated during an immune response towards the presence of non-self cells such as pathogens or cancer cells, or toward self cells in the case of autoimmune diseases. Activated immune cells are particularly recruited in the localization wherein the inflammation, triggered by the presence of non-self cells, occurs. Particular markers of immune cells of activation that can be targeted by the antigen binding domain are particularly described here after.

[0334] Preferably, the activated immune cells are selected from the group consisting of activated T cells, activated B cells, activated myeloid cells including activated macrophages and activated dendritic cells. Preferably, the activated immune cell is an activated T cell.

[0335] In an embodiment, the target of the antigen binding domain is selected from the group consisting of CD3, CD4, CD8, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD119, CD137 / 4-1BB / TNFRSF9, CD150 / SLAMF1, CD153, CD154 / CD40L, CD28, CD223 / LAG-3, CD226, CD25, CD254, CD26, CD27, CD30, CD39 / ENTPD1, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, LFA-1, TIM-1, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE / CD103, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PD-1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206 / MRC1, LTBR / CD70, TNFSF14, SLAMF7, NKG2A, 2B4, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR,

[0336] BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF18 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD74, CD165, 4-1BBL / CD137L, ICOSL / CD275, SIRPa, SIRPG, TREM2, LILRB2, Dectin-1, CLEVER1 and CD160, preferably selected from the group consisting of SIRPa, TREM2, LILRB2, CD206 / MRC1, CD163, CLEC-1A, Dectin-1, CLEVER1 and CLEC-9A."T cell" or "T lymphocytes" as used herein includes CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells, effector T cells, effector memory stem like T cells, Tumor Infiltrating Lymphocyte (TIL), anergic T cells and exhausted T cells. In a very particular aspect, the T cell is an effector T cell, an exhausted T cell, a Tumor Infiltrating Lymphocyte (TIL) or an effector memory stem like T cell. "Activated T cell" or "Activated T lymphocytes" are T cells activated by simultaneously receiving signal- 1 from T-cell recognition of antigen via the T cell receptor and signal-2 from costimulatory molecule. Markers expressed by activated T cells include but are not limited to CD137 / 4-1BB / TNFRSF9, PD-1, CTLA-4, FasL / TNFSF6, ITGAE / CD103 and OX40 / TNFRSF4.

[0337] Preferably, the term "T cell" does not include regulatory T cells (Treg), inhibitory T cells and / or senescent T cells. Regulatory T cells can be characterized by the presence of the marker CD25.

[0338] "B cells" or "B lymphocytes" as used herein include but are not limited to B-l B cells, follicular B cells, and marginal zone (MZ) B cells. "Activated B cells" or "Activated B lymphocytes" are B cells activated when the B cell binds to an antigen via its B cell Receptor. Activated B cells are particularly able to secrete immunoglobulins. Markers expressed by activated B cells include but are not limited to BCMA / TNFRSF17, CD150 and CD86.

[0339] "Neutrophils" are a type of white blood cells called granulocytes and are produced from stem cells in the bone marrow. They play an important role in innate immunity "Activated Neutrophils" are neutrophils activated upon their entry into inflammatory or infected tissue site, in response to pro- inflammatory stimuli in the tissue. Neutrophils activation is characterized by the release of granule proteins, acquisition of phagocytic capabilities, and production of NETs, all of which are designed to enhance the cells' pathogen-destruction capacity. Markers of activated neutrophils include but are not limited to CDllb, CD18, CD66b, CD177 and PRTN3. "Eosinophils" are a variety of white blood cells and one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood. "Activated Eosinophils" are eosinophils which were recruited from the blood into the tissues at sites of inflammation or infection, and received activation signal through cytokine mediation. Upon activation, eosinophils can release an array of inflammatory mediators. Markers of activated eosinophils include but are not limited to CD69, L-selectin, ICAM-1, CD44 and PSGL-1.

[0340] "Basophils" are a type of polymorphonuclear leukocyte characterized by having a nucleus with two or three lobes, and by the presence of cytoplasmic granules. "Activated Basophils" are activated by antigen crosslinking of FceRI receptor-bound IgE to undergo rapid degranulation and release their cellular contents. In addition, basophils can be activated without IgE crosslinking by inflammatory mediators such as complement factors C5a and C3a, MBP, PAF and chemokines. Markers of activated basophils include but are not limited to CD63, CD203c and CD164.

[0341] "Mast cells", also known as mastocytes or labrocytes are resident cells of connective tissue that contains many granules rich in histamine and heparin. "Activated Mast cells" are mast cells stimulated by allergens through cross-linking with immunoglobulin E receptors (e.g., FceRI), physical injury through pattern recognition receptors for damage-associated molecular patterns (DAMPs), microbial pathogens through pattern recognition receptors for pathogen-associated molecular patterns (PAMPs), and various compounds through their associated G-protein coupled or ligand-gated ion channels. Upon activation, they can selectively or quickly release mediators or inflammation-inducing components, such as histamine, heparin, cytokines, and growth factors. Markers of activated mast cells include but are not limited to CD63, CD203, c-Kit, IL-3Ra, and FCERI.

[0342] "Macrophages" are a type of white blood cells that help eliminate foreign substances by engulfing foreign materials and initiating an immune response. As used herein, this term includes for example Adipose tissue macrophages, Monocytes, Kupffer cells, Sinus histiocytes, Alveolar macrophages (dust cells), Tissue macrophages (histiocytes) leading to giant cells, Microglia, Hofbauer cells, Intraglomerular mesangial cells, Osteoclasts, Langerhans cells, Epithelioid cells, Red pulp macrophages (sinusoidal lining cells), Peritoneal macrophages, LysoMac. "Activated Macrophages" are macrophages activated either by a priming signal through IFNg followed by encountering an appropriate stimulus, such as bacterial LPS, or by direct stimulation by IL4 and / or IL13. Activated macrophages are able to kill non-self cells through phagocytosis. Markers of activated macrophages include but are not limited to TNFRSF12A / FN14 / TWEAKR.

[0343] "Dendritic cells" or "DCs" are antigen-presenting cells of the immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. As used herein, this term includes for example plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC). "Activated Dendritic cells" are dendritic cells directly activated by conserved pathogen molecules and indirectly by inflammatory mediators produced by other cell types that recognize such molecules. Markers of activated dendritic cells include but are not limited to TRAF4.

[0344] "Natural killer cel I", "NK cells" or "large granular lymphocytes (LGL)" are a type of cytotoxic lymphocyte critical to the innate immune system that contains small granules in their cytoplasm comprising proteins such as perforin and proteases known as granzymes. As used herein, this term includes natural killer cells, adaptive natural killer cells, memory natural killer cells and memory-like natural killer cells. "Activated Natural killer cell" are natural killer cells activated by a prevalence of activating receptor stimulation over inhibitory receptor stimulation. Markers of activated natural killer cells include but are not limited to NKG2A and TRAF3IP1.

[0345] The antigen binding domain of the invention particularly targets a marker of activation of an immune cell, in particular such as described here above.

[0346] Such targets are more particularly described in the Table E below.

[0347] Table E: Examples of target expressed on immune cells.

[0348] For instance, the antigen binding domain binds to a target expressed on activated T cells surface, preferably selected from the group consisting of CD101 / IGSF2, CD119, CD137 / 4-1BB / TNFRSF9, CD183 / CXCR3, CD25, CD254, CD26, CD275 / ICOSL, CD40L / CD154, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR6, FasL / TNFSF6, GITR / TNFRSF18 / CD357, GPR32, ICOS, IL18Rl / CXCRl / CD218a, ITGAE / CD103, LY108 / SlamF6, OX40 / TNFRSF4, RGS1, PD-1, LTBR / CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3 / TNFRSF6B, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12 and CD160.

[0349] For instance, the antigen binding domain binds to a target expressed on activated B cells surface, preferably selected from the group consisting of BCMA / TNFRSF17, CD150, CD86, OX40L, LOX1, TACI / TNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, IL18Rl / CXCRl / CD218a, CD74 and CD165. For instance, the antigen binding domain binds to a target expressed on activated myeloid cells surface, preferably selected from the group consisting of CD163, CD206, SIGLEC 6, TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R, SIRPa, TREM2, CLEC9A, CLEC-1A.

[0350] For instance, the antigen binding domain binds to a target expressed on activated natural killer cells surface, preferably selected from the group consisting of CST7, CXCR4, NKG2A, TRAF3IP1, CMKLR1.

[0351] Preferably, the target specifically expressed on activated immune cells surface is not an antigen of the TCR pathway (interaction between antigen presenting cells and T cells).

[0352] Preferably, the antigen binding domain binds to a target expressed on activated immune cells surface selected from the group consisting of PD-1, CD127, SIRPa, CD206 / MRC1, CD163, CLEC-9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A. In a specific aspect, the antigen binding domain has an antagonist activity on the target. In an alternative aspect, the antigen binding domain has a agonist activity on the target.

[0353] In a very specific aspect, the antigen binding domain is not an antagonist of PD-1, CD127, SIRPa, CD206 / MRC1, CD163, CLEC-9A, TREM2, LILRB2, Dectin-1, CLEVER1 or CLEC-1A. More specifically, it can be a non-antagonist and even preferably an agonist of PD-1, CD127, SIRPa, CD206 / MRC1, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 or CLEC-1A.

[0354] As used herein, the terms "Programmed Death 1", "Programmed Cell Death 1", "PD-1", "PDCD1", "PD- 1 antigen", "human PD-1", "hPD-1" and "hPD-1" are used interchangeably and refer to the Programmed Death-1 receptor, also known as CD279, and include variants and isoforms of human PD- 1, and analogs having at least one common epitope with PD-1. PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCDl gene. As an example, the amino acid sequence of a human PD-1 is disclosed under Gen Bank accession number NP_005009. PD-1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC). Accordingly, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD-1, such as PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4. Unless specified otherwise, the terms include any variant and, isoform of human PD-1 that are naturally expressed by PBMC, or that are expressed by cells transfected with a PD-1 gene.

[0355] Several anti-PD-1 are already clinically approved, and others are still in clinical developments. For instance, the anti-PD-1 antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO- 4538), OSE-279 (see WO2020 / 127366), Pidilizumab (CT-011), Cemiplimab (Libtayo), Camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (Tisleizumab), PDR001 (spartalizumab), MK-3477, SCH- 900475, PF-06801591, JNJ-63723283, Genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT- 1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), JS001 (see Si-Yang Liu et al., J. Hematol. Oncol.10:136 (2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see WO 2014 / 194302), AGEN2034 (see WO 2017 / 040790), MGA012 (see WO 2017 / 19846), or IBI308 (see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, the disclosure thereof being incorporated herein by reference), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006 / 121168, the disclosure thereof being incorporated herein by reference.

[0356] Antibodies directed against TIM-3 targeting TIM-3 are also known such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367. In some aspects, the TFM-3 antibody is as disclosed in International Patent Application Publication Nos. W02013006490, W02016 / 161270, WO 2018 / 085469, or WO 2018 / 129553, WO 2011 / 155607, U.S. 8,552,156, EP 2581113 and U.S 2014 / 044728, the disclosure thereof being incorporated herein by reference.

[0357] In some aspects, the target is SIRPa and the antigen binding domain is specific to SIRPa, preferably human SIRPa. Preferably, the antigen binding domain is an agonist of SIRPa.

[0358] As used herein, the terms "Signal-regulatory protein alpha", "SIRPa" and "SIRPa" refers to a receptortype transmembrane glycoprotein that is mammalian Immunoglobulin-like cell surface receptor for CD47. The term "anti-SIRP" refer to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and specifically binds to SIRPa, in particular to a human SIRPa, to one or both of two common variants identified, SIRPaVI and SIRPaV2. For example, the human SIRPa amino acid sequence is about 504 amino acids and has a Genbank accession number of NP_001035111.1, NP_001035112.1, NP_001317657.1, or NP_542970.1.

[0359] In such aspects, the antigen binding domain is a non-antagonist and even preferably is an agonist of SIRPa, thereby inhibiting the activation of myeloid cells. When the antigen binding domain is specific of SIRPa, the multifunctional molecule as defined herein is not for use in oncology (i.e., for the treatment of cancer). Preferably, when a construction comprises a SIPRa antigen binding domain, such as an anti-SIRPa antibody, said antigen binding domain is not an antagonist of SIRPa. In a very particular aspect, the antigen binding domain is a humanized anti-human SIPRalpha antibody or antigen-binding fragment thereof having an agonist effect on SIRPalpha.

[0360] Agonist antibodies directed against SIRPa are also known in the art, such as 6F2, for example such as disclosed in Cell Rep Med. 2023 Aug 15;4(8):101130, the disclosure thereof being incorporated herein by reference.

[0361] In some aspects, the agonist anti-SIPRa antigen binding domain comprises or consists of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ. ID NO: 138, or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion, and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ. ID NO: 139, or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion.

[0362] In some aspects, the target is CLEC-1A and the antigen binding domain is specific to CLEC-1A, preferably human CLEC-1A. Preferably, the antigen binding domain is an antagonist of CLEC-1A.

[0363] As used herein, the term "CLEC-1 A" relates to a C- type lectin-like receptor- 1 A from a mammal species, preferably a human CLEC-1 A. A reference sequence of the human CLEC-1 A corresponds to the sequence associated to the Accession number Q8NC01 Uniprot. As used herein, the term "CLEC-1 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CLEC-1. In particular, the CLEC-1 antagonist inhibits the interactions between the CLEC-1 and at least one of its ligands.

[0364] Antibodies directed against CLEC-1A are also known in the art, such as MAB1704, ABIN526589, AF1704 and ABIN526590.

[0365] In some aspects, the target is CD206 / MRC1 and the antigen binding domain is specific to CD206, preferably human CD206. Preferably, the antigen binding domain is an antagonist of CD206.

[0366] As used herein, the term " CD206 " relates to a Macrophage mannose receptor 1 from a mammal species, preferably a human CD206. A reference sequence of the human CD206 corresponds to the sequence associated to the Accession number P22897 Uniprot. As used herein, the term " CD206 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CD206. In particular, the CD206 antagonist inhibits the interactions between the CD206 and at least one of its ligands.

[0367] Antibodies directed against CD206 are also known in the art, such as PE-65155, 2A6A10, ARC0538 and CAB11192.

[0368] In some aspects, the target is CD163 and the antigen binding domain is specific to CD163, preferably human CD163. Preferably, the antigen binding domain is an antagonist of CD163.

[0369] As used herein, the term " CD163 " relates to a Scavenger receptor cysteine-rich type 1 protein M130 from a mammal species, preferably a human CD163. A reference sequence of the human CD206 corresponds to the sequence associated to the Accession number Q86VB7 Uniprot. As used herein, the term " CD163 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CD163. In particular, the CD163 antagonist inhibits the interactions between the CD163 and at least one of its ligands.

[0370] Antibodies directed against CD163 are also known in the art, such as GHI / 61, OR2805 and SQabl9148. In some aspects, the target is CLEC9A and the antigen binding domain is specific to CLEC9A, preferably human CLEC9A. Preferably, the antigen binding domain is an antagonist of CLEC9A. As used herein, the term " CLEC-9A " relates to a C- type lectin-like receptor- 1 A from a mammal species, preferably a human CLEC-9 A. A reference sequence of the human CLEC-9 A corresponds to the sequence associated to the Accession number Q6UXN8 Uniprot. As used herein, the term "CLEC-9 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CLEC-9. In particular, the CLEC-9 antagonist inhibits the interactions between the CLEC-9 and at least one of its ligands. Antibodies directed against CLEC-9A are also known in the art, such as ab222794 and 55451-1-AP.

[0371] In some aspects, the target is TREM2 and the antigen binding domain is specific to TREM2, preferably human TREM2. Preferably, the antigen binding domain is an antagonist of TREM2.

[0372] As used herein, the term " TREM2" relates to a Triggering receptor expressed on myeloid cells 2 from a mammal species, preferably a human TREM2. A reference sequence of the human TREM2 corresponds to the sequence associated to the Accession number Q9NZC2 Uniprot. As used herein, the term " TREM2 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of TREM2. In particular, the TREM2 antagonist inhibits the interactions between the TREM2 and at least one of its ligands.

[0373] Antibodies directed against TREM2 are also known in the art, such as 9H4L26, 4H42L9, TR2-H5257 and TR2-H5254.

[0374] In some aspects, the target is LILRB2 and the antigen binding domain is specific to LILRB2, preferably human LILRB2. Preferably, the antigen binding domain is an antagonist of LILRB2.

[0375] As used herein, the term " LILRB2 " relates to a Leukocyte immunoglobulin-like receptor subfamily B member 2 from a mammal species, preferably a human LILRB2. A reference sequence of the human LILRB2 corresponds to the sequence associated to the Accession number Q8N423 Uniprot. As used herein, the term " LILRB2 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of LILRB2. In particular, the LILRB2 antagonist inhibits the interactions between the LILRB2 and at least one of its ligands.

[0376] Antibodies directed against LILRB2 are also known in the art, such as JTX-8064, 10-108, 42D1 and 27D6. In some aspects, the target is Dectin-1 and the antigen binding domain is specific to Dectin-1, preferably human Dectin-1. Preferably, the antigen binding domain is an antagonist of Dectin-1.

[0377] As used herein, the term " Dectin-1 " relates to a C-type lectin domain family 7 member A from a mammal species, preferably a human Dectin-1. A reference sequence of the human Dectin- lcorresponds to the sequence associated to the Accession number Q9BXN2 Uniprot. As used herein, the term " Dectin-1 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of Dectin-1. In particular, the Dectin-1 antagonist inhibits the interactions between the Dectin-1 and at least one of its ligands.

[0378] Antibodies directed against Dectin-1 are also known in the art, such as 2A11, abl40039 and AF1859.

[0379] In some aspects, the target is CLEVER1 and the antigen binding domain is specific to CLEVER1, preferably human CLEVER1. Preferably, the antigen binding domain is an antagonist of CLEVER1.

[0380] As used herein, the term " CLEVER1 " relates to a Common lymphatic endothelial and vascular endothelial receptor-1 from a mammal species, preferably a human CLEVER1. A reference sequence of the human CLEVER1 corresponds to the sequence associated to the Accession number Q9NY15 Uniprot. As used herein, the term " CLEVER1 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CLEVER1. In particular, the CLEVER1 antagonist inhibits the interactions between the CLEVER1 and at least one of its ligands.

[0381] Antibodies directed against CLEVER1 are also known in the art, such as Bexmarilimab and FP-1305.

[0382] In another particular aspect, the target is PD-1 and the antigen binding domain of the multifunctional molecule is an antibody, a fragment or a derivative thereof or an antibody mimic that is specific to PD- 1. Then, in a particular aspect, the antigen binding domain comprised in the multifunctional molecule according to the invention is an anti-PDl antibody or antigen binding fragment thereof, preferably a human, humanized or chimeric anti-PDl antibody or antigen binding fragment thereof. Preferably, the antigen binding domain is an antagonist of PD-1. In some aspects, the anti-PD-1 antibody is Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538) or OSE-279 (such as described in WO2020 / 127366, the disclosure thereof being incorporated herein by reference).

[0383] In a very specific aspect of the present disclosure, the antigen binding domain targets PD-1 and is derived from the antibody disclosed in WO2020 / 127366, the disclosure thereof being incorporated herein by reference.

[0384] Then, in an aspect, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0385] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0386] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

[0387] - the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 60, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but position 3 of SEQ ID NO: 60;

[0388] - the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 61, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 13, 14 and 16 of SEQ ID NO: 61;

[0389] - the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 62; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 62;

[0390] - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 63, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 63;

[0391] - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 64, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

[0392] - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 65, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 65.

[0393] In another aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61 and a CDR3 of SEQ ID NO: 62; and (ii) a light chain variable region (VL) comprising a CDR1 of SEQ ID NO: 63, a CDR2 of SEQ ID NO: 64 and a CDR3 of SEQ ID NO: 65.

[0394] In one embodiment, the anti-PDl antibody or antigen binding fragment according to the invention comprises framework regions, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4 and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4.

[0395] Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0396] (i) a heavy chain variable region (VH) comprising a HFR1 of SEQ ID NO : 66, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR1 of SEQ ID NO: 60, a HFR2 of SEQ ID NO : 67, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR2 of SEQ ID NO: 61, a HFR3 of SEQ ID NO : 68, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR3 of SEQ ID NO: 62; and a HFR4 of SEQ ID NO : 69, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, and

[0397] (ii) a light chain variable region (VL) comprising a LFR1 of SEQ ID NO : 70, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR1 of SEQ ID NO: 63, a LFR2 of SEQ ID NO : 71, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR2 of SEQ ID NO: 64, a LFR3 of SEQ ID NO : 72, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR3 of SEQ ID NO: 65 and a LFR4 of SEQ ID NO : 73, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0398] In an aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0399] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence SEQ ID NO:

[0400] 74, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106 and 112 of SEQ ID NO: 74;

[0401] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO:

[0402] 75, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99 and 105 of SEQ ID NO: 74.

[0403] Preferably, the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).

[0404] In another aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0405] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 74; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof outside of the CDRs (i.e., in the framework region only);

[0406] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 75 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof outside of the CDRs (i.e., in the framework region only).

[0407] Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0408] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 74 and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 75.

[0409] In an aspect, the anti-PD-1 antigen-binding domain comprises VH, VL, CHI and a CL domain, so that the antigen binding domain is a Fab.

[0410] In such aspect, the heavy chain constant domain (CHI) comprises or consists essentially of SEQ ID NO:

[0411] 76, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Particularly, the anti-PD-1 antigen-binding domain comprises a heavy chain that comprises or consists of a VH of SEQ ID 74 and a CHI of SEQ ID NO:76.

[0412] Preferably, the light chain constant domain (CL) comprises or consists essentially of SEQ ID NO: 77 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Particularly, the anti-PD-1 antigen-binding domain comprises a light chain that comprises or consists of a VL of SEQ ID 75 and a CL of SEQ ID NO:77.

[0413] In an embodiment, the anti-PD-1 antigen-binding domain is a Fab or a Fab', a Fab or a F(ab')2 and comprises i) a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID No: 78, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and ii) a VL domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID No: 79, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Preferably, the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).

[0414] Preferably, the antigen binding domain is an anti-PD-1 Fab or F(ab')2, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID Nos: 74 and 76 respectively and ii) a chain comprising or consisting of VL and CL domains, said domains having the amino acid sequence as set forth in SEQ ID Nos: 75 and 77, respectively.

[0415] In an aspect, the antigen binding domain is an anti-PD-1 Fab or F(ab')2, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 78 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 79 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof. Preferably, the amino acids are outside of the CDRs, i.e., are in the framework regions (FR).

[0416] In an aspect, the antigen binding domain comprises an anti-PD-1 CrossMAb comprising or consisting of i) a chain comprising or consisting of VH and CL domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 74 and 77, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 75 and 76, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0417] Preferably, the antigen binding domain comprises an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID NOs: 74 and 77, respectively, and ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 75 and 76, respectively. In an aspect, the antigen binding domain is an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 80 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 81 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0418] Alternatively, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0419] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0420] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein

[0421] HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 90,

[0422] HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 91,

[0423] HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 92,

[0424] LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 93,

[0425] LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 94, and

[0426] LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 95,

[0427] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0428] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 96;

[0429] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 97.

[0430] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0431] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein

[0432] HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 98,

[0433] HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 99,

[0434] HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 100,

[0435] LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 101,

[0436] LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 102, and

[0437] LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 103,

[0438] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0439] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 104;

[0440] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO:

[0441] 105. In some aspects, the antigen binding domain comprised in the multifunctional molecule of the invention binds to TIG IT.

[0442] Antibodies directed against TIG IT are known in the art, such as BMS-986207 or AB154, BMS-986207

[0443] CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083,

[0444] CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5,

[0445] CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5,

[0446] CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3,

[0447] CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8 as disclosed in WO19232484. Anti-TIGIT antibodies are also disclosed in WO16028656, WQ16106302, WO16191643,

[0448] W017030823, W017037707, WO17053748, WO17152088, WO18033798, WO18102536,

[0449] WQ18102746, WQ18160704, W018200430, WO18204363, W019023504, WO19062832,

[0450] WO19129221, WO19129261, WO19137548, WO19152574, WO19154415, WO19168382 and WO19215728, the disclosure thereof being incorporated herein by reference.

[0451] In some aspects, the anti-TIGIT antigen binding domain comprises or consists of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 133, or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion, and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 134, or a variant thereof comprising 1, 2, 3, 4 or 5 amino acid modifications, preferably selected from the group consisting of substitution, addition or deletion.

[0452] Preferably, the anti-TIGIT binding domain comprises or consists of:

[0453] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 133 and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 134.

[0454] Preferably, the anti-TIGIT binding domain is a bivalent antibody comprising or consisting of:

[0455] (a) a first heavy chain, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 120,

[0456] (b) a second heavy chain, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 121, 5, 50 and 5, from N-terminal to C-terminal;

[0457] (c) two light chains, comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 122. In some aspects, the antigen binding domain is an agonist of PD-1, LAG-3, TIG IT or SIRPa.

[0458] In some aspects, the antigen binding domain is of an agonist antibody selected from the group consisting of PD-1, LAG-3, BTLA or VISTA, for example such as described in Lowell et al., Front Immunol. 2025 Mar 26;16:1566869, the disclosure of hich being incorporated by reference. In some aspects, the antigen binding domain is of an agonist anti-PD-1 antibody, preferably selected from the group consisting of Peresolimab, Rosnilimab, CC-90006, PT627, MB151, RTX-002.

[0459] In some aspects, the antigen binding domain is of an agonist anti-LAG3 antibody, preferably IMP761.

[0460] Fc domain

[0461] In a particular aspect of the disclosure, the antigen binding domain as described above may be associated with antibody constant regions, in particular from IgA, IgM, IgE, IgD or IgG such as IgGl, lgG2, lgG3, lgG4, preferably IgGl, lgG2, or lgG4. Preferably, the antigen binding domain comprises an IgG Fc region, preferably an IgGl, lgG2, or lgG4 Fc region. Preferably, the Fc domain includes all or a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from a human or humanized IgGl, lgG2, lgG3 or lgG4, preferably from a human or humanized IgGl, lgG2, or lgG4.

[0462] The molecules envisioned herein optionally comprise a Fc domain.

[0463] As used herein, the terms "fragment crystallizable region" "Fc region" or "Fc domain" are interchangeable and refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors. The Fc region or domain is typically composed of two domains, optionally identical, derived from the second and third constant domains of the antibody's two heavy chains (i.e., CH2 and CH3 domains). Portion of the Fc domain refers to the CH2 or the CH3 domain. Optionally, the Fc region or domain may optionally comprise all or a portion of the hinge region between CHI and CH2. Accordingly, the Fc domain may comprise the hinge, the CH2 domain and the CH3 domain. Optionally, the Fc domain is that from IgGl, lgG2, lgG3 or lgG4, optionally with IgGl hinge-CH2-CH3 and lgG4 hinge-CH2-CH3.

[0464] The Fc domain of the multifunctional molecule can form together with a part of the antigen binding domain a heavy chain of an IgG immunoglobulin. Indeed, when the antigen binding domain is a Fab, the multifunctional molecule may comprise one heavy chain, including the variable heavy chain (VH), CHI, hinge, CH2 and CH3 domains. However, the multifunctional molecule may also have other structures such as scFv, or diabody.

[0465] The Fc domain can be from a heavy chain constant domain of a human immunoglobulin heavy chain, for example, IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the multifunctional molecule comprises an IgGl or an lgG4 heavy chain constant domain.

[0466] Preferably, the Fc domain comprises CH2 and CH3 domains. Optionally, it can include all or a portion of the hinge region, the CH2 domain and / or the CH3 domain. In some aspects, the CH2 and / or a CH3 domains are derived from a human lgG4 or IgGl heavy chain.

[0467] Specifically, the domain derived from lgG4 or IgGl heavy chain is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the lgG4 or IgGl heavy chain or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0468] Preferably, the Fc domain includes all or a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, lgG2, lgG3, lgG4, or other classes. Preferably, the hinge region is derived from human IgGl, lgG2, lgG3, lgG4. The hinge region derived from IgGl, lgG2, lgG3, lgG4 heavy chain is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with the IgGl, lgG2, lgG3, lgG4 heavy chain or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0469] More preferably, the hinge region is derived from a human or humanized IgGl or lgG4 heavy chain. Particularly, the hinge region has a sequence of SEQ ID NO: 89 or is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity thereto or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0470] The IgGl hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines permit efficient and consistent disulfide bonding formation between Fc portions. Therefore, a preferred hinge region of the present invention is derived from IgGl, more preferably from human IgGl. In some aspects, the first cysteine within the human IgGl hinge region is mutated to another amino acid, preferably serine. For instance, the sequence including CHI, CH2 and CH3 based on an IgGl is disclosed in SEQ ID NO: 83 including a N298A substitution.

[0471] The hinge region of lgG4 is known to form interchain disulfide bonds inefficiently. However, a suitable hinge region for the present invention can be derived from the lgG4 hinge region, preferably containing a mutation that enhances correct formation of disulfide bonds between heavy chain-derived moieties (Angal S, et al. (1993) Mol. Immunol., 30:105-8). More preferably, the hinge region is derived from a human lgG4 heavy chain. For instance, the sequence including CHI, CH2 and CH3 based on an lgG4 is disclosed in SEQ ID NO: 82 including a S228P substitution.

[0472] Specifically, the heavy chain constant domains (CH1+CH2+CH3) comprise or consist of the sequence as set forth in SEQ ID NO: 82 or 83 or is a variant thereof having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of identity with SEQ ID NO: 82 or 83 or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof. Preferably, the mutations are outside the CDRs, i.e., are in the framework regions.

[0473] The multifunctional molecule comprises a dimeric Fc domain. Accordingly, two monomers comprise each one a Fc chain, the Fc chains being able to form a dimeric Fc domain. The dimeric Fc domain can be an homodimer, each Fc monomer being identical or essentially identical. Alternatively, the dimeric Fc domain can be a heterodimer, each Fc monomer being different and complementary in order to promote the formation of the heterodimeric Fc domain. More specifically, the Fc domain is a heterodimeric Fc domain. Heterodimeric Fc domains are made by altering the amino acid sequence of each monomer. The heterodimeric Fc domains rely on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and / or allow for ease of purification of heterodimers over the homodimers. There are a number of mechanisms that can be used to generate the heterodimers of the present invention. In addition, as will be appreciated by those in the art, these mechanisms can be combined to ensure high heterodimerization. Thus, amino acid variants that lead to the production of heterodimers are referred to as "heterodimerization variants". Heterodimerization variants can include steric variants (e.g., the "knobs and holes" or "skew" variants described below and the "charge pairs" variants described below) as well as "pi variants", which allows purification of homodimers away from heterodimers. WO2014 / 145806, hereby incorporated by reference in its entirety, discloses useful mechanisms for heterodimerization include "knobs and holes", "electrostatic steering" or "charge pairs", pi variants, and general additional Fc variants. See also, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, Merchant et al., Nature Biotech. 16:677 (1998), all of which are hereby incorporated by reference in their entirety. For "electrostatic steering" see Gunasekaran et al., J. Biol. Chem. 285(25): 19637 (2010), hereby incorporated by reference in its entirety. For pi variants, see US 2012 / 0149876 hereby incorporated by reference in its entirety.

[0474] Then, in a preferred aspect, the heterodimeric Fc domain comprises a first Fc chain and a complementary second Fc chain based on the "knobs and holes" technology. For instance, the first Fc chain is a "knob" or K chain, meaning that it comprises the substitution characterizing a knob chain, and the second Fc chain is a "hole" or H chain, meaning that it comprises the substitution characterizing a hole chain. And vice versa, the first Fc chain is a "hole" or H chain, meaning that it comprises the substitution characterizing a hole chain, and the second Fc chain is a "knob" or K chain, meaning that it comprises the substitution characterizing a knob chain. In a preferred aspect, the first Fc chain is a "hole" or H chain and the second Fc chain is a "knob" or K chain.

[0475] Optionally, the heterodimeric Fc domain may comprise one heterodimeric Fc chain which comprises the substitutions as shown in the following Table F and the other heterodimeric Fc chain comprising the substitutions as shown in the following Table F.

[0476] Table F (the numbering being according to EU index)

[0477] In a preferred aspect, one Fc chain is a "hole" or H chain and comprises the substitutions T366S / L368A / Y407V / Y349C and one Fc chain is a "knob" or K chain and comprises the substitutions T366W / S354C.

[0478] In a yet preferred embodiment, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) - a peptide linker and a single cytokine, variant or fragment thereof; said peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine and being a "masking" peptide linker as defined herein; or

[0479] - two peptide linkers and a single cytokine, variant or fragment thereof; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is linked to the C-terminus of the cytokine, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein; or - one peptide linker and a single cytokine, variant or fragment thereof; said peptide linker being linked to the C-terminus of the cytokine and being a "masking" peptide linker as defined herein wherein the first Fc chain comprises or consists of a "knob" chain and the second Fc chain comprises or consists of a "hole" chain.

[0480] Optionally, the Fc chain may further comprise additional substitutions.

[0481] In particular, for multifunctional molecules that target cell-surface molecules, especially those on immune cells, abrogating effector functions may be required. Engineering Fc regions may also be desired to either reduce or increase the effector function of the multifunctional molecules.

[0482] In certain aspects, amino acid modifications may be introduced into the Fc region to generate an Fc region variant. In certain aspects, the Fc region variant possesses some, but not all, effector functions. Such multifunctional molecules may be useful, for example, in applications in which the half-life of the antibody in vivo is important, yet certain effector functions are unnecessary or deleterious. Numerous substitutions or substitutions or deletions with altered effector function are known in the art.

[0483] In one aspect, the constant region of the Fc domain contains a mutation that reduces affinity for an Fc receptor or reduces Fc effector function. For example, the constant region can contain a mutation that eliminates the glycosylation site within the constant region of an IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates the glycosylation site within the CH2 domain.

[0484] In a particular aspect, the Fc domain is modified to increase the binding to FcRn, thereby increasing the half-life of the multifunctional molecule. In another aspect or additional aspect, the Fc domain is modified to decrease the binding to FcyR, thereby reducing ADCC or CDC, or to increase the binding to FcyR, thereby increasing ADCC or CDC.

[0485] The alteration of amino acids near the junction of the Fc portion and the non-Fc portion can dramatically increase the serum half-life of the Fc fusion protein as shown in WO 01 / 58957. Accordingly, the junction region of a protein or polypeptide of the present invention can contain alterations that, relative to the naturally-occurring sequences of an immunoglobulin heavy chain and erythropoietin, preferably lie within about 10 amino acids of the junction point. These amino acid changes can cause an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is replaced. Preferably, the C-terminal lysine of an IgG sequence is replaced with a non-lysine amino acid, such as alanine or leucine, to further increase serum half-life.

[0486] In one embodiment, the constant region of the Fc domain has one of the mutations described in the Table G below, or any combination thereof. Table G: Suitable human engineered Fc domain of an antibody, numbering of residues in the heavy chain constant region is according to EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63,

[0487] 78-85 (1969); www.imgt.Org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs)

[0488] In a particular aspect, the multifunctional molecule comprises a human IgGl heavy chain constant domain or an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q / M428L; M252Y / S254T / T256E + H433K / N434F; E233P / L234V / L235A / G236A + A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q311; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; P329G; N297A + M252Y / S254T / T256E; K322A, K444A, K444A, K444E, K444D, K444G, K444S , K444L, K444Q, K444N, K444I, K444P, K444V, K444T, L234A / L235A / P329G, M428L, L309D, Q311H, N434S, M428L + N434S (LS) and L309D + Q311H + N434S (DHS), preferably selected from the group consisting of N297A optionally in combination with M252Y / S254T / T256E, and L234A / L235A or L234A / L235A / P329G. Preferably, the substitution or combination of substitutions selected from the group consisting of T250Q / M428L; M252Y / S254T / T256E + H433K / N434F; E233P / L234V / L235A / G236A + A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q311; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A + M252Y / S254T / T256E; K322A and K444A, preferably selected from the group consisting of N297A optionally in combination with M252Y / S254T / T256E, and L234A / L235A.

[0489] In a particular aspect, the multifunctional molecule comprises a human IgGl heavy chain constant domain or an IgGl Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q / M428L; M252Y / S254T / T256E + H433K / N434F; E233P / L234V / L235A / G236A + A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q311; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; P329G; N297A + M252Y / S254T / T256E; K322A,K444A, K444E, K444D, K444G, K444S, M428L, L309D, Q311H, N434S, M428L + N434S and L309D + Q311H + N434S, preferably selected from the group consisting of N297A optionally in combination with M252Y / S254T / T256E, and L234A / L235A optionally with P329G.

[0490] The multifunctional molecule comprising a human IgGl heavy chain constant domain or an IgGl Fc domain with the combination of substitutions L234A / L235A / P329G greatly reduces or altogether suppresses ADCC, ADCP and / or CDC caused by said multifunctional molecule, thus reducing nonspecific cytotoxicity.

[0491] In another aspect, the multifunctional molecule comprises a human lgG4 heavy chain constant domain or a human lgG4 Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A / L235A, P329G, S228P + M252Y / S254T / T256E and K444A. Even more preferably, the multifunctional molecule, comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4. In another aspect, the multifunctional molecule comprises a human lgG4 heavy chain constant domain or a human lgG4 Fc domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A / L235A; L234A / L235A / P329G, P329G, S228P + M252Y / S254T / T256E, K444A K444E, K444D, K444G and K444S. Even more preferably, the multifunctional molecule, preferably the multifunctional molecule according to the invention comprises an lgG4 Fc-region with a S228P that stabilizes the lgG4.

[0492] As mentioned herein the and "+" refer to mutations that are cumulative. Thus, by the mutation S228P + M252Y / S254T / T256E, it is meant the following mutations: S228P, M252Y, S254T and T256E.

[0493] The multifunctional molecule comprising a human lgG4 heavy chain constant domain or an lgG4 Fc domain with the substitution P329G reduces ADCC and / or CDC caused by said multifunctional molecule, thus reducing nonspecific cytotoxicity.

[0494] All subclass of Human IgG carries a C-terminal lysine residue of the antibody heavy chain (K444) that are susceptible to be cleaved off in circulation. This cleavage in the blood may compromise or decrease the bioactivity of the multifunctional molecule by releasing the linked immune-stimulating moiety to the multifunctional molecule. To circumvent this issue, K444 amino acid in the IgG domain can be substituted by another amino acid to reduce proteolytic cleavage, a mutation commonly used for antibodies. Then, in one aspect, the multifunctional molecule comprises at least one further amino acid substitution consisting of K444A, K444E, K444D, K444G, K444L, K444Q, K444N, K444I, K444P, K444V, K444T or K444S, preferentially K444A or K444L. Particularly, K444 amino acid in the IgG domain can be substituted by an alanine or a lysine to reduce proteolytic cleavage, a mutation commonly used for antibodies. Then, in one aspect, the multifunctional molecule comprises at least one further amino acid substitution consisting of K444A or K444L.

[0495] Optionally, the multifunctional molecule comprises an additional cysteine residue at the C-terminal domain of the Fc domain to create an additional disulfide bond and potentially restrict the flexibility of the multifunctional molecule.

[0496] In one particular aspect, the multifunctional molecule according to the invention comprises a heterodimer of Fc domains that comprises the "knob into holes" modifications such as described above. Preferably, such Fc domains are IgGl or lgG4 Fc domain such as described above, even more preferably an IgGl Fc domain comprising the mutation N297A such as disclosed above.

[0497] For instance, the first Fc chain is a "knob" or K chain and comprises the substitutions T366W / S354C and optionally N297A and the second Fc chain is a "hole" or H chain and comprises the substitutions T366S / L368A / Y407V / Y349C and optionally N297A. Preferably, the first Fc chain is a "knob" or K chain and comprises the substitutions T366W / S354C and N297A and the second Fc chain is a "hole" or H chain and comprises the substitutions T366S / L368A / Y407V / Y349C and N297A. More particularly, the knob Fc chain may comprise or consist in SEQ ID NO: 84 and / or the hole Fc chain may comprise or consist in SEQ ID NO: 85.

[0498] Accordingly, an object of the present invention relates to a polypeptide comprising, from the N- terminal to the C-terminal, an antigen binding domain or a part thereof, a Fc chain (knob or hole Fc chain), preferably the hole-chain of the Fc domain, and either:

[0499] - a peptide linker and a single cytokine, variant or fragment thereof; said peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine and being a "masking" peptide linker as defined herein; or

[0500] - two peptide linkers and a single cytokine, variant or fragment thereof; wherein a first linker is covalently linked to the N-terminal end of the cytokine and a second linker is linked to the C-terminal end of the cytokine, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein.

[0501] More specifically, the antigen binding domain according to the invention is linked to the knob-chain and / or the hole chain of the heterodimeric Fc domain.

[0502] In a first aspect, the multifunctional molecule comprises or consists of:

[0503] - a first entity comprising or consisting of a first Fc chain which is a knob Fc chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine

[0504] - a second entity comprising i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; iii) a "masking" peptide linker as defined herein, and iv) a cytokine.

[0505] In a second aspect, the multifunctional molecule comprises or consists of:

[0506] - a first entity comprising or consisting of a first Fc chain which is a knob Fc chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine

[0507] - a second entity comprising or consisting of i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; optionally, a peptide spacer, iii)) a cytokine, and iv) a "masking" peptide linker as defined herein.

[0508] In a third aspect, the multifunctional molecule comprises or consists of:

[0509] - a first entity comprising or consisting of a first Fc chain which is a knob chain; said Fc chain being preferably devoid of an antigen binding domain and of a cytokine

[0510] - a second entity comprising i) an antigen binding domain, ii) a second Fc chain complementary to the first Fc chain which is a hole Fc chain, the first (knob) and second (hole) Fc chain forming together a Fc domain; iii) a first peptide linker, iv) a cytokine and v) a second peptide linker, at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein, preferably both being a "masking" peptide linker as defined herein. Preferably, in the multifunctional molecule of the invention comprising a Fc domain, particularly a heterodimeric Fc domain, the peptide linker(s) and the cytokine are linked to the hole Fc chain, preferably to the C-terminal end of the hole Fc chain.

[0511] The multifunctional molecule may comprise one or more spacers, especially peptide spacers, particularly between the antigen binding domain and the Fc domain. Such a spacers may be useful to prevent steric hindrances. The spacer is usually 3-44 amino acid residues in length. Preferably, the spacer has 3-30 amino acid residues. In some aspects, the spacer has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acid residues.

[0512] The spacer sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the spacer is preferably non-immunogenic in the subject to which the multifunctional molecule is administered. One useful group of spacer sequences are derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Other examples are poly-alanine sequences. Further preferred examples of spacer sequences are Gly / Ser linkers of different length including (Gly4Ser)4(SEQ ID NO: 107), (Gly4Ser)3 (SEQ ID NO: 106), (Gly4Ser)? (SEQ ID NO: 108), Gly4Ser, Gly3Ser, Gly3, Gly2Ser and (Gly3Ser2)3(SEQ ID NO: 109).

[0513] In some embodiments, when the multifunctional molecule comprises only one peptide linker at the C- terminus of the cytokine, the multifunctional molecule may further comprise a peptide spacer between the antigen binding domain or the Fc domain and the cytokine. In addition, if the cytokine is a dimer, a peptide spacer can be used to covalently linked the two subunits of the cytokine.

[0514] In some embodiments, the multifunctional molecule does not comprise a glycine-serine linker, preferably such as defined above, especially that links the cytokine to the antigen binding domain, typically between the Fc domain and the cytokine.

[0515] In some embodiments, the multifunctional molecule of the invention does not comprise a cleavable linker, in particular comprising a protease cleavage site, typically a metalloprotease cleavage site. of multifunctional molecules

[0516] Examples of multifunctional molecules of the invention are provided below.

[0517] In some aspects, the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures A, B, Cl, C2, C3, C4, C5, C6, C7, C8 and D of Figure 1C.

[0518] In some aspects, the structure of the multifunctional molecule is selected from the structure disclosed in Figure 1A, 4A and from the structures Cl, C2, C3, C4, C5, C6, C7 and C8 of Figure 1C.

[0519] In some aspect, the structure and / or sequence of the multifunctional molecule is selected from the structure and / or sequence disclosed in Table 5 of the Example section, when a peptide linker of the invention is comprised, at the exception of the constructions comprising an antagonist SIRPa, particularly comprising an amino acid sequence as set forth in SEQ ID NO: 127, 128, 129 and 135, these constructions being only for illustrative purposes.

[0520] In some aspects, the multifunctional molecule comprises one of the following arrangements, said arrangements being described from the N-terminus to the C-terminus: antigen binding domain heavy chain (VH or VH+CH1)- optionally a Fc chain - "masking" linker

[0521] - cytokine - "masking" linker, antigen binding domain light chain (VL or VL+CL) -"masking" linker - cytokine - "masking" linker, antigen binding domain heavy chain (VH or VH+CH1)- optionally a Fc chain - "masking" linker - cytokine, antigen binding domain light chain- "masking" linker - cytokine, antigen binding domain heavy chain(VH or VH+CH1) — optionally a Fc chain - cytokine - "masking" linker, antigen binding domain light chain (VL or VL+CL)- - cytokine - "masking" linker, antigen binding domain heavy chain(VH or VH+CH1) — optionally a Fc chain - "masking" linker

[0522] - cytokine - linker, antigen binding domain light chain (VL or VL+CL)- -"masking" linker - cytokine - linker, antigen binding domain heavy chain (VH or VH+CH1)- - optionally a Fc chain - linker - cytokine

[0523] - "masking" linker, and an antigen binding domain light chain (VL or VL+CL)- - linker - cytokine - "masking" linker.

[0524] These arrangements may be combined i) with each other as long as the antibody comprises a VH and a VL, ii) with a Fc domain devoid of a cytokine; iii) ii) with a Fc domain devoid of a cytokine and of antigen binding domain or iv) with an antigen binding domain light chain or heavy chain that is not linked to a cytokine.

[0525] In some preferred aspects, the multifunctional molecule according to the invention comprises or consists of an antibody (monovalent or bivalent) - a first masking linker - a cytokine - a second masking linker, preferably this structure being read from N-terminal to C-terminal. Especially, the C- terminal end of the Fc domain of the antibody, preferably only the hole Fc chain, is covalently linked to the N-terminal end of the first peptide linker.

[0526] In some preferred aspects, the multifunctional molecule according to the invention comprises or consists of an antibody (monovalent or bivalent), preferably an anti-SIRPa, an anti-PDl or anti-TIGIT antibody - a first EEEEEEEEEEEEEEE linker (SEQ ID NO: 5) - a cytokine, preferably selected from the group consisting of IL-2, IL-15, IL-10 and IL-18 - a second EEEEEEEEEEEEEEE linker (SEQ ID NO: 5), preferably this structure being read from N-terminal to C-terminal. Especially, the C-terminal end of the Fc domain of the antibody, preferably only the hole Fc chain, is covalently linked to N-terminal end of the first peptide linker.

[0527] In some aspects, the multifunctional molecule according to the invention comprises or consists of:

[0528] - an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A or a fragment thereof; in particular as described above;

[0529] - one, two, three or four, preferably two, cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal and / or C-terminal end of the antibody;

[0530] - one to eight, preferably one to four, peptide linker(s) as described above covalently linked to the N- terminal and / or C-terminal end of the cytokine(s), wherein at least one peptide linker per cytokine is a "masking" peptide linker as defined herein.

[0531] In some aspects, the multifunctional molecule according to the invention comprises or consists of:

[0532] - an anti-SIPRa antibody or a fragment thereof; in particular as described above;

[0533] - one, two, three or four, preferably two, cytokine(s), variant(s) or fragment(s) thereof covalently linked to the N-terminal and / or C-terminal end of the antibody;

[0534] - one to eight, preferably one to four, peptide linker(s) as described above covalently linked to the N- terminal and / or C-terminal end of the cytokine(s), wherein at least one peptide linker per cytokine is a "masking" peptide linker as defined herein.

[0535] In some aspects, the multifunctional molecule comprises or consists of:

[0536] - an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A or a fragment thereof; in particular as described above;

[0537] - a single cytokine or a variant or fragment thereof covalently linked to the C-terminal end of the antibody;

[0538] - one or two peptide linker(s) covalently linked to the N-terminal and / or C-terminal end of the cytokine(s) as described above, wherein at least one peptide linker is a "masking" peptide linker as defined herein and preferably both.

[0539] Preferably, the structure of the multifunctional molecule, from N-terminal to C-terminal is selected from the group consisting of: anti-SIRPa antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; anti-SIRPa antigen binding domain, preferably antibody - first peptide linker-cytokine; anti-PDl antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; anti-PDl antigen binding domain, preferably antibody - first peptide linker-cytokine; anti-TIG IT antigen binding domain, preferably antibody - first peptide linker-cytokine- second peptide linker; and anti- TIG IT antigen binding domain, preferably antibody - first peptide linker-cytokine.

[0540] In the above structures, the antibody can be bivalent or monovalent. The peptide linker and / or cytokine are covalently linked to the C-terminal end of the Fc chain of the antibody, preferably the hole Fc chain.

[0541] Preferably, in said multifunctional molecules, the cytokine is an anti-inflammatory cytokine, preferably IL-2, IL-10, IL-35, IL-37 and IL-38 or a variant thereof, more preferably IL-2 or IL-10.

[0542] Preferably, in said multifunctional molecules, the first peptide linker is E15 (EEEEEEEEEEEEEEE (SEQ ID NO: 5). More preferably, the first and the second peptide linkers are E15 (EEEEEEEEEEEEEEE (SEQ ID NO: 5).

[0543] Preferably, in the multifunctional molecule, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),

[0544] NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46). More specifically, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),

[0545] DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),

[0546] NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43). In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).

[0547] In an even more particular aspect, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), most preferably is EEEEEEEEEEEEEEE (SEQ ID NO: 5).

[0548] In some aspect, in the multifunctional molecule, the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), I I I I I I I I I I I I TTH (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), T l I I I I I I I I I I I I HTTTTT (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I TTH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).

[0549] In some aspects, the multifunctional molecule comprises or consists of: a Fc domain, preferably an heterodimeric Fc domain comprising a "knob" chain and a "hole" chain, a single antigen binding domain covalently linked to the N-terminus of the Fc domain, said antigen binding domain comprising or consisting of a VH and a VL domains (e.g., Fab) of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A such as described above, a single cytokine covalently linked to the C-terminus of the Fc domain, and one or two peptide linker(s), preferably two peptide linkers, covalently linked to N-terminus and / or C-terminus of the cytokine, as described above, wherein at least one peptide linker is a "masking" peptide linker as defined herein and preferably both. Preferably, the antigen binding domain and the cytokine are on the same Fc chain. Especially, the antigen binding domain and the cytokine are on the "hole" Fc chain.

[0550] In some aspects, the molecule comprises or consists of: a) a first entity comprising a first Fc chain ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A such as described above, ii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) either

[0551] - one "masking" peptide linker as defined herein and a single cytokine; said "masking" peptide linker being covalently linked to the N-terminus or C-terminus of the cytokine; or

[0552] - two peptide linkers and a single cytokine; wherein a first linker is covalently linked to the N-terminus of the cytokine and a second linker is covalently linked to the C-terminus of the cytokine, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; or

[0553] - one "masking" peptide linker, one peptide spacer and a single cytokine; wherein a peptide spacer is covalently linked to the N-terminus of the cytokine and a "masking" peptide linker is covalently linked to the C-terminus of the cytokine; or one "masking" peptide linker, one peptide spacer and a single cytokine; wherein a peptide spacer is covalently linked to the C-terminus of the cytokine and a "masking" peptide linker is covalently linked to the N-terminus of the cytokine;.

[0554] In the multifunctional molecule, the cytokine is an anti-inflammatory cytokine, preferably IL-2, IL-10, IL-35, IL-37 or IL-38 or a variant thereof such as described above.

[0555] Preferably, in the multifunctional molecule, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),

[0556] NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46). More specifically, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32),

[0557] DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37),

[0558] NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43). In a very particular aspect, the "masking" peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), and ETETETETETETETE (SEQ ID NO: 44).

[0559] In a even more particular aspect, the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), and EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48).

[0560] In some aspect, in the multifunctional molecule, the second or additional peptide linker(s) comprise(s), essentially consist(s) of or consist(s) of an amino acids sequence selected from the group consisting PPPPSPPPPSPPPPS (SEQ ID NO: 1), AAAAHAAAAAAAAAA (SEQ ID NO: 3), I I I I I I I I I I I I TTH (SEQ ID NO: 4), AAAAHAAAAAAAAAAAAAAA (SEQ ID NO: 6), AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7), AAAAAAAAAAAAAAA (SEQ ID NO: 25), T l I I I I I I I I I I I I I I I I I I (SEQ ID NO: 8), (SEQ ID NO: 9), PPPPSPPPPS (SEQ ID NO: 23), PPPPSPPPPSPPPPSPPPPS (SEQ ID NO: 24), I I I I I I I I I H (SEQ ID NO: 27), I I I I I I I I I HT I I I I I I TTH (SEQ ID NO: 28), AAAAAAAAAA (SEQ ID NO: 29) and AAAAHAAAAAAAAAAHAAAA (SEQ ID NO: 30).

[0561] Preferably, in the multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.

[0562] In some aspects, the molecule comprises or consists of: a) a first entity comprising a first Fc chain ; said Fc chain being devoid of an antigen binding domain and of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A such as described herein, ii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-10, IL-35, IL-37, IL-38 or a variant or fragment thereof such as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine; the first and / or second peptide linker(s) preferably consist of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49).

[0563] Preferably, in the multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.

[0564] In some aspects, the multifunctional molecule comprises or consists of: a) a first chain comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two "masking" peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-2 and consists of SEQ ID NOs : 50; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.

[0565] Preferably, in such multifunctional molecule, the first Fc chain is a knob chain and the second Fc chain is a hole chain.

[0566] In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an antibody directed against a target expressed by a myeloid cell as described herein (i.e. CD163, CD206, CLEC-9A, TREM2), ii) a first Fc chain; said Fc chain being devoid of a cytokine ; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and a VL domains (e.g., Fab) of an antibody directed against a target expressed by a T cell as described herein (i.e. PD-1), ii) a second Fc chain complementary to the first Fc entity, the first and second Fc entities forming together a Fc domain; and iii) two peptide linkers and a single cytokine which is a cytokine, preferably IL-2, IL-10, IL-35, IL-37, IL-38 or a variant thereof, in particular as described herein; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine; the first and / or second peptide linker(s) preferably consist of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49).

[0567] In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A, preferably an anti-SIRPa or CLEC1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-2 and consists of SEQ ID NO : 50; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.

[0568] In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A, preferably an anti-SIRPa or CLEC1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-10 and consists of SEQ ID NO : 51; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.

[0569] In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A, preferably an anti-SIRPa or CLEC1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-37 and consists of SEQ ID NO : 54; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.

[0570] In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A, preferably an anti-SIRPa or CLEC1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-38 and consists of SEQ ID NO : 55; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.

[0571] In some aspects, the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; b) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A, preferably an anti-SIRPa or CLEC1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-35 comprising EBI3 subunit of SEQ ID NO : 52 and p53 subunit of SEQ ID NO: 53; wherein a first peptide linker is covalently linked to the N-terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine.

[0572] In some aspects, the multifunctional molecule comprises or consists of: c) a first entity comprising a first Fc chain of SEQ ID NO :84, said Fc chain being devoid of an antigen binding domain and of a cytokine; d) a second entity comprising i) a single antigen binding domain, said antigen binding domain comprising a VH and VL of an antibody that specifically binds to a target selected from the group consisting of SIRPa, CD206, CD163, CLEC9A, TREM2, LILRB2, Dectin-1, CLEVER1 and CLEC-1A, preferably an anti-SIRPa or CLEC1 antibody such as described herein ii) a second Fc chain of SEQ ID NO : 85 complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iii) two peptide linkers, both selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), and EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), and a single cytokine which is IL-12 comprising a p35 subunit (IL-12A) of SEQ ID NO : 56 and p40 subunit (IL-12B) of SEQ ID NO: 57; wherein a first peptide linker is covalently linked to the N- terminus of the cytokine and a second peptide linker is covalently linked to the C-terminus of the cytokine. In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), preferably EEEEEEEEEEEEEEE (SEQ ID NO: 5), (v) IL2 or IL10 or a variant thereof, preferably comprising or consisting of SEQ ID NO: 50, 51, 112 or 117 and vi) a second peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), preferably EEEEEEEEEEEEEEE (SEQ ID NO: 5), a second heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a second Fc chain complementary to the first Fc chain, the second Fc chain being preferably a knob chain such as comprising or consisting of SEQ ID NO: 84; said second heavy chain being preferably devoid of an IL-2 molecule or cytokine, and a first and second light chains of an anti-PD-1 antibody preferably comprising or consisting of SEQ ID NO: 79.

[0573] In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-PDl VH and CHI domain of an antibody, preferably comprising or consisting of SEQ ID NO: 78, ii) optionally a hinge domain, preferably comprising or consisting of SEQ ID NO: 89, iii) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), (v) IL2 or I LIO or a variant thereof, preferably such as described herein in particular in SEQ ID NO: 50, 51, 112 and 117, and vi) a second peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49); a second heavy chain comprising or consisting of a second Fc chain complementary to the first Fc chain, the second Fc chain being preferably a knob chain such as comprising or consisting of SEQ ID NO: 84; said second heavy chain being preferably devoid of a cytokine, and of an anti-PD-1 antigen binding domain; and a first and second light chains of an anti-PD-1 antibody preferably comprising or consisting of SEQ ID NO: 79.

[0574] In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of SEQ ID NO: 84; a second heavy chain comprising or consisting of an amino acid sequence comprising or consisting of SEQ ID NO: 88-5-(50, 51, 112 or 117)-5, from N terminus to C terminus; and a first and second light chains comprising or consisting of SEQ ID NO: 79.

[0575] In some aspects, the bifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of from N-terminal to C-terminal : i) an anti-TIGIT VH domain preferably comprising or consisting of SEQ ID NO: 133, ii) optionally a CHI domain of an antibody , iii) optionally a hinge domain, iv) a first Fc chain, preferably a Fc hole chain such as comprising or consisting of SEQ ID NO: 85, iv) a first peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), (v) a cytokine, preferably selected from the group consisting of IL-2 or IL-10 or a variant thereof, in particular such as disclosed in SEQ ID NO: 50, 51, 112 or 117, and vi) a second peptide linker of the invention, preferably consisting of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48),

[0576] EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49); a second heavy chain comprising or consisting of a second Fc chain complementary to the first Fc chain, the second Fc chain being preferably a knob chain such as comprising or consisting of SEQ ID NO: 84; said second heavy chain being preferably devoid of a cytokine, and of an anti-PD-1 antigen binding domain; and a first and second light chains of an anti-TIGIT antibody preferably comprising or consisting of

[0577] SEQ ID NO: 134.

[0578] In some aspects, the multifunctional molecule of the invention comprises or consists of: a first heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 120 a second heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 121-5-(50, 51, 112 or 117)-5 from N-terminus to C-terminus; and a first and second light chains comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 122.

[0579] Preparation of multifunctional molecule

[0580] To produce a multifunctional molecule according to the invention, in particular by mammalian cells, nucleic acid sequences or group of nucleic acid sequences coding for the multifunctional molecule are subcloned into one or more expression vectors. Such vectors are generally used to transfect mammalian cells. General techniques for producing molecules comprising antibody sequences are described in Coligan et al. (eds.), Current protocols in immunology, at pp. 10.19.1-10.19.11 (Wiley Interscience 1992), the contents of which are hereby incorporated by reference and in "Antibody engineering: a practical guide" from W. H. Freeman and Company (1992), in which commentary relevant to production of molecules is dispersed throughout the respective texts.

[0581] Preferably, the multifunctional molecule is a polypeptide or protein produced as a recombinant protein.

[0582] Generally, such method comprises the following steps of:

[0583] (1) transfecting or transforming appropriate host cells with the polynucleotide(s) encoding the recombinant multifunctional molecule of the invention or the vector containing the polynucleotide(s);

[0584] (2) culturing the host cells in an appropriate medium; and

[0585] (3) optionally isolating or purifying the multifunctional molecule from the medium or host cells.

[0586] The invention further relates to a nucleic acid encoding a multifunctional molecule as disclosed above, a vector, preferably an expression vector, comprising the nucleic acid of the invention, a genetically engineered host cell transformed with the vector of the invention or directly with the sequence encoding the recombinant multifunctional molecule, and a method for producing the multifunctional molecule of the invention by recombinant techniques.

[0587] The nucleic acid, the vector and the host cells are more particularly described hereafter.

[0588] Nucleic acid molecules encoding the molecules or multifunctional molecules of the present invention. Recombinant Expression Vectors and Host Cells comprising such

[0589] Nucleic acid sequence The invention also relates to an isolated nucleic acid molecule encoding the multifunctional molecule as defined above or to a group of isolated nucleic acid molecules encoding the multifunctional molecule as defined above. Nucleic acid encoding the multifunctional molecule disclosed herein can be amplified by any techniques known in the art, such as PCR. Such nucleic acids may be readily isolated and sequenced using conventional procedures.

[0590] In a particular aspect, the nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:

[0591] - a first nucleic acid encoding a polypeptide comprising, from N to C-terminus i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer iii) a first Fc chain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;

[0592] - a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; and

[0593] - a third nucleic acid molecule encoding the light chain of the antibody.

[0594] In a particular aspect, the nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:

[0595] - a first nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody ii) optionally a peptide spacer and iii) a first Fc chain;

[0596] - a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both;

[0597] - a third nucleic acid molecule encoding the light chain of the antibody.

[0598] In a particular aspect, the nucleic acid molecules encoding the multifunctional molecule as defined herein comprises:

[0599] - a first nucleic acid molecule encoding a first Fc chain; and

[0600] - a second nucleic acid molecule encoding a polypeptide comprising, from N to C-terminus, i) a heavy chain (VH) of an antibody, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain so that the first and second Fc chain form together a Fc domain; iv) a first peptide linker, v) a cytokine and vi) a second peptide linker, wherein at least one of the first and second peptide linkers is a "masking" peptide linker as defined herein and preferably both; - a third nucleic acid molecule encoding the light chain of the antigen-binding domain.

[0601] In one embodiment, the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule.

[0602] Vectors

[0603] In another aspect, the invention relates to a vector comprising the nucleic acid molecule or the group of nucleic acid molecules as defined above.

[0604] As used herein, a "vector" is a nucleic acid molecule used as a vehicle to transfer genetic material into a cell. The term "vector" encompasses plasmids, viruses, cosmids and artificial chromosomes. In general, engineered vectors comprise an origin of replication, a multicloning site and a selectable marker. The vector itself is generally a nucleotide sequence, commonly a DNA sequence, that comprises an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Modern vectors may encompass additional features besides the transgene insert and a backbone: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have control sequences.

[0605] The nucleic acid molecule encoding the multifunctional molecule, the fusion protein, can be cloned into a vector by those skilled in the art, and then transformed into host cells. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The methods known to the artisans in the art can be used to construct an expression vector containing the nucleic acid sequence of the multifunctional molecule, variant described herein and appropriate regulatory components for transcription / translation.

[0606] Accordingly, the present invention also provides a recombinant vector, which comprises a nucleic acid molecule encoding the multifunctional molecule according to the present invention. In one preferred aspect, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally at least one drug-resistance gene for screening. The expression vector may further comprise a ribosome -binding site for initiating the translation, transcription terminator and the like.

[0607] Suitable expression vectors typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination / polyadenylation sequence.

[0608] An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants.

[0609] Lipid based nanoparticles

[0610] In another aspect, the invention relates to a lipid-based nanoparticle comprising the multifunctional molecule of the invention or a nucleic acid sequence or vector encoding such.

[0611] The lipid-based nanoparticle according to the invention is particularly formulated either as a liposome or a lipid nanoparticle (LNP), especially a lipid nanoparticle comprising a mixture of lipids.

[0612] The lipid-based nanoparticle also encompasses similar nanoparticles such as but not limited to micelles and nano-emulsions.

[0613] Lipid-based nanoparticles typically comprise helper lipid, sterol and / or PEG lipid components along with the mRNA of interest. The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a lipid-based nanoparticle may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements.

[0614] In a preferred embodiment, the lipid-based nanoparticles is a lipid nanoparticle comprising one or more ionizable or cationic lipid(s), one or more helper lipid(s), one or more sterol(s), and / or one or more polyethylene glycol (PEG)-modified lipid(s).

[0615] Host cells

[0616] In another aspect, the invention relates to a host cell comprising a vector or a nucleic acid molecule or group of nucleic acid molecules as defined above, for example for multifunctional molecule production purposes.

[0617] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be or has been recipient of vectors, exogenous nucleic acid molecules, and polynucleotides encoding the multifunctional molecule according to the present invention. The term "host cell" is also intended to include progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, rabbit, macaque or human.

[0618] Suitable hosts cells are especially eukaryotic hosts cells which provide suitable post-translational modifications such as glycosylation. Preferably, such suitable eukaryotic host cell may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe; insect cell such as Mythimna separate; plant cell such as tobacco, and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells and COS cells.

[0619] Preferably, the host cell of the present invention is selected from the group consisting of CHO cell, COS cell, NSO cell, and HEK cell. Then host cells stably or transiently express the multifunctional molecule according to the present invention. Such expression methods are known by the man skilled in the art.

[0620] A method of production of the multifunctional molecule is also provided herein. The method comprises culturing a host cell comprising a nucleic acid encoding the multifunctional molecule as provided above, under conditions suitable for its expression, and optionally recovering the multifunctional molecule from the host cell (or host cell culture medium). Particularly, for recombinant production of a multifunctional molecule, nucleic acid encoding a multifunctional molecule, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. The molecules or multifunctional molecules are then isolated and / or purified by any methods known in the art. These methods include, but are not limited to, conventional renaturation treatment, treatment by protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, supercentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and the combination thereof. As described, for example, by Coligan, multifunctional molecule isolation techniques may particularly include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography and ion exchange chromatography. Protein A preferably is used to isolate the molecules or multifunctional molecules of the invention.

[0621] Pharmaceutical Compositions and uses thereof

[0622] The present invention also relates to a pharmaceutical composition comprising a multifunctional molecule as described herein, the isolated nucleic acid molecule, the group of isolated nucleic acid molecules, the vector and / or the host cells as described hereabove, preferably as the active ingredient or compound. The formulations can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers, excipients, salts, anti-oxidant and / or stabilizers which do not deleteriously interact with the multifunctional molecule of the invention, nucleic acid, vector and / or host cell of the invention and does not impart any undesired toxicological effects. Optionally, the pharmaceutical composition may further comprise an additional therapeutic agent.

[0623] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active agents, such as comprising a multifunctional molecule according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism.

[0624] In one aspect, a "composition" typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. An "acceptable vehicle" or "acceptable carrier" as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0625] Compositions of the present invention can be in a form suitable for any conventional route of administration or use. Particularly, the pharmaceutical composition according to the invention can be formulated for any conventional route of administration including a topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. To facilitate administration, the multifunctional molecule as described herein can be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005).

[0626] The pharmaceutical composition may be prepared by mixing a multifunctional molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, anti-oxidant, and / or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, anti-oxidant, and / or stabilizers are well known in the art and have been for example described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0627] To facilitate delivery, any of the multifunctional molecules or its encoding nucleic acids can be conjugated with a chaperon agent. The chaperon agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipid. It can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polypeptide.

[0628] Pharmaceutical compositions according to the invention may be formulated to release the active ingredients (e.g., the multifunctional molecule of the invention) substantially immediately upon administration or at any predetermined time or time period after administration. The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Means known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[0629] It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer. Pharmaceutical composition typically is sterile and stable under the conditions of manufacture and storage. Prevention of presence of microorganisms may be ensured both by sterilization procedures (for example by microfiltration), and / or by the inclusion of various antibacterial and antifungal agents. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.

[0630] Use in the treatment of a disease

[0631] The multifunctional molecules, nucleic acids, vectors, host cells, compositions and methods of the present invention have numerous in vitro and in vivo utilities and applications. Particularly, any of multifunctional molecules, nucleic acid molecules, group of nucleic acid molecules, vectors, host cells or pharmaceutical composition provided herein may be used in therapeutic methods and / or for therapeutic purposes, in particular for the treatment of diseases or as a medicament or vaccine.

[0632] The term "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease or of the symptoms of the disease. It designates both a curative treatment and / or a prophylactic treatment of a disease. A curative treatment is defined as a treatment resulting in cure or a treatment alleviating, improving and / or eliminating, reducing and / or stabilizing a disease or the symptoms of a disease or the suffering that it causes directly or indirectly. A prophylactic treatment comprises both a treatment resulting in the prevention of a disease and a treatment reducing and / or delaying the progression and / or the incidence of a disease or the risk of its occurrence. In certain aspects, such a term refers to the improvement or eradication of a disease, a disorder, an infection or symptoms associated with it. In other aspects, this term refers to minimizing the spread or the worsening of inflammatory diseases or autoimmune diseases. Treatments according to the present invention do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a composition including one or more active agents to a subject who has a disorder / disease.

[0633] As used herein, the term "medicament" refers to any substance or composition with curative or preventive properties against disorders or diseases.

[0634] The present invention particularly relates to a multifunctional molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition comprising such for use in the treatment of a disease in a subject and / or for use as a medicament or vaccine. It also relates to the use of a multifunctional molecule as described herein; a nucleic acid or a vector encoding such, or a pharmaceutical composition comprising such for treating a disease and / or disorder in a subject. It also concerns the use of a multifunctional molecule, a nucleic acid, group of nucleic acid molecules or a vector encoding such, or a pharmaceutical composition comprising such as disclosed herein in the manufacture of a medicament for treating a disease and / or disorder in a subject, such as inflammatory diseases or autoimmune diseases. Examples of diseases to be treated are more particularly described hereafter. Finally, it relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule to the subject, or a nucleic acid or group of nucleic acid molecules or a vector encoding such. In one aspect, the treatment method comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of a multifunctional molecule, nucleic acid, vector or pharmaceutical composition as described herein.

[0635] A subject in need of a treatment may be a human having, at risk for, or suspected of having a disease. Such a patient can be identified by routine medical examination.

[0636] The invention particularly relates to a method of treatment of an inflammatory diseases or disorders or autoimmune diseases or disorders in a subject in need thereof comprising administering to said subject an effective amount of a multifunctional molecule or pharmaceutical composition as defined above.

[0637] In another aspect, the multifunctional molecules disclosed herein can be administered to a subject, e.g., in vivo, to decrease immunity, preferably in order to treat a disorder and / or disease. Accordingly, in one aspect, the invention provides a method of decreasing an immune response in a subject comprising administering to the subject a multifunctional molecule, nucleic acid, vector or pharmaceutical composition of the invention such that the immune response in the subject is decreased. The multifunctional molecule or pharmaceutical composition can be used to decrease immune responses such as T cell activation in a subject in need of a treatment.

[0638] The invention particularly provides a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutic effective amount of any of the multifunctional molecules, nucleic acid, vector or pharmaceutical composition comprising such described herein, such that an immune response in the subject is decreased. Auto immune disease

[0639] The multifunctional molecules or pharmaceutical compositions of the invention can be used for the treatment of patients affected by an autoimmune diseases such as diabetes, NASH, in particular type I diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, celiac disease, vasculitis, myasthenia gravis; infection diseases such as sepsis, severe viral indications with severe inflammatory conditions, such as coronavirus (e.g.COVID-19), peritonitis; degenerative diseases; wound healing disorders, dry eye syndrome.

[0640] Fibrosis (notably lung and hepatic fibrosis) and ANCA (Anti neutrophil Cytoplasmic Autoantibodies) pathologies (vasculitis) are in particular concerned.

[0641] Inflammatory disease

[0642] The multifunctional molecules or pharmaceutical compositions of the invention can be used for the treatment of patients affected by a disease wherein the resolution of inflammation is delayed or disrupted, and / or a disease selected from the group of inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory pulmonary diseases (e.g asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, cutaneous inflammation.

[0643] Combined therapies

[0644] In some embodiment, the multifunctional molecule or the pharmaceutical composition according to the invention may be used in combination with another therapeutic agent or therapy, in particular for the treatment of autoimmune diseases or autoimmune diseases or disorders.

[0645] The present invention also relates to a method for treating a disease, such as an autoimmune diseases or a disease wherein the resolution of inflammation is delayed or disrupted, and / or an inflammatory diseases, in a subject, comprising administering to said subject a therapeutically effective amount of the lipid multifunctional molecule or the pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent or therapy.

[0646] Specific examples of additional or second therapeutic agents are provided in W02021 / 069709.

[0647] Accordingly, also provided herein are combined therapies with any of the multifunctional molecules or pharmaceutical composition comprising such, as described herein and a suitable second agent, for the treatment of a disease or disorder.

[0648] In an aspect, the multifunctional molecule and the second agent can be present in a unique pharmaceutical composition. Alternatively, the terms "combination therapy" or "combined therapy", as used herein, embrace administration of these two agents (e.g., a multifunctional molecule as described herein and an additional or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., a multifunctional molecule) can be administered intramuscularly, and an additional therapeutic agent (e.g., an anti-inflammatory agent, or an immune modulator) can be administered intravenously. Alternatively, an agent of the combination selected may be administered by intravenous injection while the other agents of the combination may be administered intramuscularly.

[0649] In an aspect, the additional therapeutic agent can be selected in the non-exhaustive list comprising alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic, antiproliferative, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukaemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, inhibitors of inhibitors of apoptosis proteins (lAPs), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids, plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

[0650] Combination therapy could also rely on the combination of the administration of the multifunctional molecule according to the invention or the pharmaceutical composition comprising such with surgery.

[0651] Subject, regimen and administration

[0652] The present invention relates to a multifunctional molecule as disclosed herein, a nucleic acid molecule or group of nucleic acid molecules or a vector encoding such, a host cell or a pharmaceutical composition for use as a medicament or for use in the treatment of a disease or for administration in a subject in need thereof. It also relates to a method for treating a disease or a disorder in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a multifunctional molecule to a subject.

[0653] "An effective amount" or a "therapeutic effective amount" as used herein refers to the amount of active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The "effective amount" will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0654] The subject to treat may be a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old or of at least 70 years old.

[0655] In a particular aspect, the subject can be immunosuppressed (e.g., the subject has an auto-immune disease or has a transplant).

[0656] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the multifunctional molecule or the pharmaceutical composition as disclosed herein to a subject, depending upon the type of diseases to be treated or the site of the disease e.g., administered orally, parenterally, enterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Preferably, the multifunctional molecule or the pharmaceutical composition as disclosed herein is administered via subcutaneous, intra-cutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, intra-thecal, intra-lesion, and intracranial injection or infusion techniques.

[0657] The form of the pharmaceutical compositions, the route of administration and the dose of administration of the pharmaceutical composition or the multifunctional molecule according to the invention can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, size, sex, and / or general physical condition. The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired. Kits

[0658] Any of the multifunctional molecules or compositions described herein may be included in a kit provided by the present invention. The present disclosure particularly provides kits for use in decreasing immune responses and / or treating diseases or disorders (e.g., auto-immune or inflammatory diseases or disorders)

[0659] In the context of the present invention, the term "kit" means two or more components (one of which corresponding to the multifunctional molecule, the nucleic acid molecule, the vector or the cell of the invention) packaged in a container, recipient or otherwise. A kit can hence be described as a set of products and / or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit. The kits of this invention are in suitable packaging.

[0660] Particularly, a kit according to the invention may comprise:

[0661] - a multifunctional molecule as defined above,

[0662] - a nucleic acid molecule or a group of nucleic acid molecules encoding said multifunctional molecule,

[0663] - a vector comprising said nucleic acid molecule or group of nucleic acid molecules, and / or

[0664] - a cell comprising said vector or nucleic acid molecule or group of nucleic acid molecules.

[0665] The kit may thus include, in suitable container means, the pharmaceutical composition, fusion proteins or multifunctional molecules, and / or host cells of the present invention, and / or vectors encoding the nucleic acid molecules of the present invention, and / or nucleic acid molecules or related reagents of the present invention. In some embodiments, means of taking a sample from an individual and / or of assaying the sample may be provided. The compositions comprised in the kit according to the invention may particularly be formulated into a syringe compatible composition.

[0666] In some embodiments, the kit further includes an additional agent for treating auto-immune or inflammatory diseases or disorders, and the additional agent may be combined with the pharmaceutical composition, fusion proteins or multifunctional molecules, and / or host cells of the present invention, and / or vectors encoding the nucleic acid molecules of the present invention, and / or nucleic acid molecules, or other components of the kit of the present invention or may be provided separately in the kit. Particularly, the kit described herein may include one or more additional therapeutic agents such as those described in the "Combined Therapy" described hereabove. The kit(s) may be tailored to a particular auto-immune or inflammatory diseases or disorders for an individual and comprise respective second therapies suitable for the treatment of auto-immune or inflammatory diseases or disorders for the individual as described hereabove.

[0667] The instructions related to the use of the multifunctional molecule or pharmaceutical composition described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, means for reconstituting the multifunctional molecule and / or means for diluting the multifunctional molecule of the invention. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual).

[0668] All the references cited in this description are incorporated by reference in the present application. Others features and advantages of the invention will become clearer in the following figures and examples which are given for purposes of illustration and not by way of limitation.

[0669] BRIEF DESCRIPTION OF THE FIGURES

[0670] Figure 1: Schematic representation of an embodiment of the multifunctional molecule according to the invention (A), cis demasking and cis activation technology of the bifunctional molecule constructed with optimized linker (B) and example of bifunctional antibody format designed with the linkers (C). Examples of bifunctional antibody format designed with the linker are: A: antibody with a single antigen binding domain fused in C-terminal of the Hole Fc chain to a cytokine and a peptide linker, B: antibody with a single antigen biding domain fused in C-terminal of the Hole Fc chain to a peptide linker and a cytokine, Cl : antibody fused in C-terminal of the Hole Fc chain to a first peptide linker, a cytokine and a second peptide linker; C2 antibody fused in C-terminal of one light chain to a first peptide linker, a cytokine and a second peptide linker; C3 : Monovalent Fab and a Fc domain, wherein a first peptide linker, a cytokine and a second peptide linker is fused to the N-terminal of one chain of the Fc domain; C4 : Bivalent scFv linked to a Fc domain, a first peptide linker, a cytokine and a second peptide linker is fused to the C-terminal of one chain of the Fc domain; C5 : antibody fused in C-terminal of each Fc chain to a first peptide linker, a cytokine and a second peptide linker; C6: antibody fused in N-terminal of each light chain to a first peptide linker, a cytokine and a second peptide linker; C7: antibody fused in C-terminal of the Holde Fc chain to a first peptide linker, a first cytokine subunit, a second peptide linker, a second cytokine subunit and a third linker; C8 : antibody fused in C-terminal of each heavy and light chain to a first peptide linker, a cytokine and a second peptide linker, D: antibody fused in C-terminal of the Hole Fc chain to a peptide linker and a cytokine. These structures are given for purposes of illustration and not by way of limitation.

[0671] Figure 2: Optimized linkers allow a cytokine masking effect on PD-l-cell lines while inducing a specific cis-potentiation on PD-1+ cell lines. Jurkat cells expressing PD-1- CD122+CD132+ ( I L2Rbg) (A) or PD- 1+CD122+CD132+ (I L2Rbg) (B) were incubated with IL2 wild-type cytokine (•), with anti PD-1-(G4S)3- IL2wt (0) and anti-PDl-E15-IL2wt-E15 ( ▼ ). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30 min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.

[0672] Figure 3: Optimized linkers in multifunctional molecules with wild-type I L12 allow to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines. (A) Constructs used in this figure (B) Splenocytes T cells from wild-type C57bl6 mice (human PD-1 negative, hPD-1-) or human PD-1 Knock in C57bl6 mice (human PD-1+, hPD-l+) were isolated and sorted prior pSTAT4 bioassay to evaluate IL-12 signaling following treatment with Anti PD-1-IL12 treatments. T cells were activated during 4 days with agonist anti-msCD3 (145-2C11) and anti-msCD28 (37.51) then treated with mouse I L12 wild-type cytokine (• dashed line), with anti PD-l-(G4S)3-mslL12p40-(G4S)3- mslL12p35 (• plain line), anti-PDl-E15-mslL12p40-E15-mslL12p35-E15 (■) hPDl+ and hPDl- T cells were separately incubated with the different constructions at escalating doses for 15min at 37°C and percentage of pSTAT4 signal was quantified by flow cytometry. (C) A ratio of EC50 pSTA4 value on PD- 1- negative T cells vs PD1 + T cells reported on Figure. A mouse IL-12 cytokine was used for the design of the construction. N= 2 independent experiments

[0673] Figure 4: Trans-activation assay on PD-1 presentation by antigen coating or expressing cells alone or in co-culture on PD-l-cells lines and PD-1+ cells lines. (A) pStat5 signaling measured on Jurkat cells PD-1- CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone ( ▼ ) or pre-incubated on PD1 coating (□) and Jurkat cells PD-1+CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone (•) (B) pStat5 signaling measured on Jurkat cells PD-1- CD122+CD132+ added to anti-PDl-E15-IL15wt-E15 alone ( ▼ ) or pre-incubated on U937 PD1+ cells (□) and Jurkat cells PD-1+CD122+CD132+ added to anti-PDl-E15- IL15wt-E15 alone (•). (C) Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye incubated with IL15wt alone (o) or anti-PDl-E15-IL15wt-E15 (V) and Jurkat PD- 1+ IL15Rbg+ cells incubated with IL15wt alone (•) or anti-PDl-E15-IL15wt-E15 ( ▼ ) at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (D) EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells during co-culture assay and independently cells assays with IL15 alone or with anti-PDl-E15- IL15wt-E15.

[0674] Figure 5: Study assay of one or two optimized linker in construct to mask cytokine activity on PD-l- cells and allow cis-demasking and cis-potentiation of PD-1+ cells lines. (A) Schematic representation of the different constructions used in (B) and (C) is depicted in the figure: Anti PD-1 construction is monovalent and IL15wt cytokine was fused with either a (G4S)3 linker, two optimized E15 linker or a single optimized E15 linker, or without any linker with an optimized E15 linker fused to the cytokine. Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD- 1+CD122+CD132+ (IL15Rbg) (B) were incubated with anti PD-l-(G4S)3-IL15wt (square), anti-PDl-E15- IL15wt-E15 (black circle), anti-PDl-E15-IL15wt (stars) or anti-PDl-IL15wt-E15 (triangle). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (C) pSTAT5 bioassay on Jurkat PD-1 negative CD122+CD132+ (I LISRbg) was performed with escalating dose of monovalent anti PD-l-IL15wt molecules constructed with one (G4S)3 flexible linker ((G4S)3-IL15n), Two (G4S)3 flexible linker ((G4S)3-IL15-(G4S)3) (♦), two (EAAAK)3 rigid linker ((EAAAK)3) ( ▼ ) or the optimized E15 linker(E15-IL15wt-E15 (♦).

[0675] Figure 6: Peptide linkers of different amino-acid sequences tested on PD-l-cells and PD-1+ cells lines.

[0676] Jurkat cells expressing PD-1- CD122+CD132+ (IL15Rbg) (A) or Jurkat cells expressing PD- 1+CD122+CD132+ (IL15Rbg) (B) were incubated with IL15 wild-type cytokine (grey dot), anti PD-1- (G4S)3-IL15wt (square), isotype-E15-IL15wt-E15 (white dot), anti-PDl-E15-IL15wt-E15 (grey diamond), anti-PDl-D15-IL15wt-D15 (triangle) or anti-PDl-N15-IL15wt-N15 (cross). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (C) EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells for each compound. (D) Experiment was repeated on primary T cells sorted from human PBMC (n=3) donors, either on naive cells (37% PD1+ cells) cells stimulated with PHA ) (75% PD1+ cells) or cells stimulated twice with CD3 / CD28 (100% PD1+ cells) . Constructions tested were designed with monovalent anti PD-1 antibody (Anti PD-1*1) and a wild type IL-15 fused optimized linkers (E15-15, D15-D15, N15-N15) Figure 7: Peptide linkers comprising different amino-acid sequences tested on PD-l-cells and PD-1+ cells lines. Jurkat cells expressing PD1+ CD122+ or expressing PD1+ and CD122+ but blocked with lmg / mL of an anti-PDl were incubated with I L15 alone, anti-PD-1 (G4S)3-IL15, or anti-PDl X15-IL15- X15, where X is a combination of different proportion of amino acid Glu (E) and Asp (D) (Fig. 7A), Glu and Thr (T) (Fig 7B) Glu and Ala (A) (Fig 7C)) or Glu and Lys (K) (Fig. 7D). Each PD-1- (PD1+ cells saturated with anti-PD-1) and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.

[0677] Figure 8: Different length of optimized linkers tested to mask cytokine activity on PD-l-cells and allow cis-demasking and cis-potentiation of PD-1+ cell lines. Jurkat cells expressing PD-1-CD122+ (A) or expressing PD-1+ CD122+cells (B) were incubated with anti PD-1-(G4S)3-IL15 (white square); anti- PD1-E6-IL15-E6 (light grey diamond), anti-PDl-E10-IL15-E10 (triangle), anti-PDl-E15-IL15-E15 (grey circle), anti-PDl-E20-IL15-E20 (grey diamond) or anti-PDl-E25-IL15-E20 (black square). Each PD-1- and PD-1+ cells were respectively incubated with the different constructions at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population.

[0678] Figure 9: Binding PD1 assay of the anti PD-1 bifunctional molecule with optimized linker or conventional linker. Binding ELISA assay was performed on PD1 coating with incubation at different concentration of either anti PD-1-(G4S)3-IL15 (0) or anti-PDl-E15-IL15-E15 ( ▲ ) for 2h at 37°C. Antibodies were detected with peroxidase-labelled anti-human IgG polyclonal antibody and revealed by TMB. Optical Density (DO) was measured at 450nm by spectrophotometer (Tecan).

[0679] Figure 10: Optimized linkers are capable of masking and demasking cytokine with one or multiple valences of anti PD-1. (A) Co-culture assay with ratio 1:1 of Jurkat PD-1- IL15Rbg+ cells stained with CellTrackerGreen dye (dotted lines) and Jurkat PD-1+ IL15Rbg+ cells (solid lines) were incubated with a monovalent anti-PDl-E15-IL15-E15 (triangle), a bivalent anti-PDl-E15-IL15-E15 (diamond) or an isotype-E15-IL15-E15 (circle) at escalating doses for 30min at 37°C and Median Fluorescent Intensity (MFI) of pStat5 was quantified by flow cytometry for each PD-1 + and PD-1- cell population. (B) EC50 values (nM) of pStat5 activation in PD-1- or PD1+ cells during co-culture assay and independently cells assays.

[0680] Figure 11: Anti PD-1 E15 IL2 E15 enhances safety as evaluated in vivo in toxicity mouse model. C57bl6 mice were intravenously injected with one or multiple doses of anti PD-1 E15 IL2 wild type E15 (black) or anti PD1 (G4S)3 IL2 wild type (white) (2mg / kg) n=3 to 4 per group. Weight was evaluated every day until day 3 and data normalized at the day 0 prior injection of the drug and statistical analysis performed* p<0.05 (A) One dose injected vs (B) multiple injections (every day).

[0681] Figure 12: Optimized linker (E15) masks activity of IL-10 cytokine on human T and B lymphocytes SIRPa negative cell lines compared to a (G4S)3 conventional flexible linker (A) Constructions used in the experiment (B) SIRPa expression on human HPB-ALL (T cell line), RAJI and RAMOS (B cell lines) using flow cytometry. (C) pSTAT3 activity measurement of human cell lines using flow cytometry after stimulation with escalating doses of human IL10 cytokine alone (naked recombinant cytokine), the monovalent Anti SIRPa / IL-10 molecule constructed with (G4S)3 or E15 linker (SEQ ID NO: 84; 128+106+51; 129 or SEQ ID 84; 128+5+51; 129, respectively ). pSTAT3 percentage of positive cells was quantify after permeabilization and intranuclear staining (n= 1 to 2 independent experiments).

[0682] Figure 13: Optimized linker (E15) masks activity of IL-10 cytokine on primary naive B / T cells and shows higher CIS activity on SIRPa+ versus SIRPa- compared to (G4S)3 conventional flexible linker (A) Constructions used in the experiment (B) SIRPa expression on naive human PBMCs using flow cytometry. (C) pSTAT3 activity measurement into naive human T cells, B cells and Monocytes using flow cytometry after stimulation with escalating doses of the monovalent Anti SIRPa / IL-10 molecule constructed with (G4S)3 or E15 linker (SEQ ID NO: 84; 128+106+51; 129 or SEQ ID NO: 84; 128+5+51; 129, respectively). pSTAT3 percentage of positive cells was quantified after permeabilization and intranuclear staining, and was evaluated after gating into CD3+, CD20 and CD14+ respectively (n= 5-8 donors tested). (D) EC50 was determined with both constructions compared recombinant human I L10 for each population. Figure 14: Optimized linker (E15) masks activity of bivalent IL-10 cytokine construction on primary naive B / T cells and higher CIS activity on SIRPa+ myeloid cells compared to IL-10 non targeted cytokine (A) Constructions used for the experiments (B) pSTAT3 activity measurement of naive human T cells, B cells and Monocytes using flow cytometry after stimulation with escalating doses of the monovalent Anti SIRPa / IL-10 molecule constructed with (bivalent E15 linker, SEQ ID NO: 135+5+51; 129) pSTAT3 percentage of positive cells was quantified after permeabilization and intranuclear staining, and was evaluated after gating CD3+, CD20 and CD14+ respectively (n= 4-8 donors tested).

[0683] Figure 15: Optimized linker allows both cis-demasking and cis-potentiation on SIRPa+ monocytes. Monocytes were pre-saturated with anti-SIRPa antibody (50pg / mL, same clone as immunocytokine construction) to have no access to SIRPa (representing SIRPa- non targeted cells) pSTAT3 activity on SIRPa+ (• Black) and SIRPa- (• Grey) naive monocytes was assessed with an escalating doses for 15min at 37°C. Percentage of pStat3 was quantified by flow cytometry after intracellular staining (anti pSTAT3 AF647) in monocytes (confirmed on n=l-9). CIS activity fold-change on SIRPa neg versus SIRP+ cells was evaluated using EC50 ratio in each cell type and reported on the graph for each construction: (A) Monovalent anti-SIRPa was fused to IL-10 with E15 linker (SED ID NO: 84; 128+5+51; 129).. (B) Bivalent anti-SIRPa was fused to IL-10 with a E15 linker (SED ID NO: 135+5+51; 129).

[0684] Figure 16: Anti SIRPa E15 I LIO efficiently inhibits macrophages activation and secretion of pro- inflammatory cytokines in a whole blood bioassay. (A &B) Whole blood from healthy volunteers was stimulated over-night with 100 ng / mL LPS and treated with escalating doses of a monovalent Anti SIRPa / IL-10 molecule constructed with (G4S)3 or E15 linker or a bivalent Anti-SIRPa with bivalent E15- IL10 (SEQ ID NO: 84; 128+106+51; 129, SEQ ID 84; 128+5+51; 129 and SEQ ID NO: 135+5+51; 129 respectively). Human IL-6; TNFa and I FNg were assessed by ELISA and results are expressed as relative to secretion with LPS only. (A) Optimized linker combined with SIRPa targeting allows similar antiinflammatory properties as recombinant human IL-10 (B) Construction with bivalent IL-10 with E15 linker also presented a high anti-inflammatory properties (confirmed on n=3-9 donors).

[0685] Figure 17: Anti SIRPa E15 IL-10 molecule suppresses secretion of proinflammatory IL-6 cytokine by myeloid cells isolated from ulcerative colitis patient. Total leukocytes were depleted in red blood cells and isolated from whole blood of ulcerative colitis patient, then stimulated over-night with 100 ng / mL LPS and treated with escalating doses of the monovalent Anti SIRPa / IL-10 constructed with E15 linker. Human IL-6 was assessed by ELISA. Optimized linker combined with SIRPa targeting allows better antiinflammatory properties (n=l)

[0686] Figure 18: Optimized linker (E15) masks and inhibits pro-inflammatory activity of IL-10 cytokine on activated CD8+ T cells while the same construction with a (G4S)3 conventional flexible linker stimulates proinflammatory function of T cells (A) protocol of the experiment. Naive human CD8+ T cells were isolated from buffy coat and stimulated 3 days with CD3 / CD28. Second activation was conducted with CD3 and with escalating doses of recombinant human IL-10 or the monovalent Anti SIRPa / l L-10 molecule constructed with (G4S)3 or E15 linker or bivalent Anti-SIRPa with bivalent E15- IL10 (SEQ ID NO: 84; 128+106+51; 129 ; SEQ ID NO: 84; 128+5+51; 129 and SEQ ID No: 135+5+51; 129, respectively) for 3 days. Cells were washed and stimulated for 4 hours with CD3 for supernatant analysis. Granzyme B and I FNg were dosed by ELISA (n= 3-4 donors tested)

[0687] Figure 19: Optimized linker (E15) masks activity of IL-10 cytokine on mouse primary naive SIRPa negative cells and shows higher CIS activity on SIRPa+ versus SIRPa- compared to (G4S)3 conventional flexible linker (A) Constructions used in this experiments : bivalent Anti mouse SIRPa / IL- 10 constructed with (G4S)3 or E15 linker (SEQ ID NO: 130; 131+106+51; 132 and SEQ ID NO: 130; 131+5+51; 132 ; 6F2 clone). (B) SIRPa expression on naive mouse splenocytes (lb & T cells) or from bone marrow (macrophages) using flow cytometry. (C) pSTAT3 activity measurement of naive mouse T cells, B cells (from spleen) and Macrophages (from bone marrow) using flow cytometry after stimulation with escalating doses of the molecules. MFI of pSTAT3 was quantify after permeabilization and intranuclear staining, and was evaluated after gating CD3+, B220+ and CD14+ respectively (n= 5-8 donors tested). (D) EC50 was determined with both constructions compared recombinant human I L10 in each population.

[0688] Figure 20: Anti SIRPa-E15 IL-10 allows a specific CIS activation of mouse SIRPa+ cells using Raw cell lines. (A) RAW264.7 mouse macrophage cell line was used. Mouse SIRPa expression was confirmed by flow cytometry. (B) pSTAT3 activity on SIRPa targeted cells(* Black) and without SIRPa targeting (• Grey) on mouse macrophages was assessed with an escalating doses for 15min at 37°C. pStat3 activation was quantified by flow cytometry after intracellular staining (anti pSTAT3 AF647) and expressed in relative to the control without treatment (confirmed on n=6). (C) CIS activity of the molecule as evaluated by EC50 ratio of SIRPa targeting vs no targeting....

Claims

1. CLAIMS1. A multifunctional molecule comprising: an antigen binding domain comprising or consisting of an antibody or an antigen binding fragment or derivative thereof; a cytokine or a variant fragment thereof having an anti-inflammatory effect covalently linked to the N-terminal and / or C-terminal end of the antigen binding domain, and a non-cleavable peptide linker covalently linked to the N-terminal and / or C-terminal end of the cytokine, wherein the peptide linker is of 5 to 30 amino acids in length and consists of- at least 20% acidic or amidic amino acids independently selected from the group consisting of E, D and N,- up to 20% basic amino acids selected from the group consisting of H, K and R provided that the peptide comprises at least 3 times more acidic or amidic amino acids than basic amino acids, and- at least 70, 80 or 90% of the remaining amino acids being independently selected from the group consisting of G, P, A, V, S, and T, preferably selected from the group consisting of A, T and S.

2. The multifunctional molecule according to claim 1, wherein the peptide linker is a masking peptide linker.

3. The multifunctional molecule according to claim 1 or 2, wherein the peptide linker consists of an amino acid sequence selected from the group consisting of a sequence having at least 50% of polar, amidic and acidic amino acids independently selected in the group consisting of E, D, N, Q, T and S and having between 0% and 80% of amino acids being independently selected from the group consisting of G, P, A, V, S, and T; a sequence having between 20% and 100% of amino acids independently selected from the group consisting of E, D and N; a sequence having 5 to 30 consecutive amino acids E; a sequence having 5 to 30 consecutive amino acids D; and a sequence having 5 to 30 consecutive amino acids N.

4. The multifunctional molecule according to claim 1 or 2, wherein said peptide linker comprises:- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids selected independently from the group consisting of E, D and N; or- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being E; or- 3, 4, 5, &, , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being D; or- 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 amino acids being N.

5. The multifunctional molecule according to claim 1 or 2, wherein the peptide linker comprises, essentially consists of or consists of an amino acid sequence selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), EEEEEEEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 49), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34),DDDDDDDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 35), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39),NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 40), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEEAEEEEA (SEQ ID NO: 16), EEEEAEEEEAEEEEA (SEQ ID NO: 17), EEEEAEEEEAEEEEAEEEEA (SEQ ID NO: 18), EAEAEAEAEAEAEAE (SEQ ID NO: 45), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), ETTTTETTTTETTTT (SEQ ID NO: 43), and EEEEKEEEEKEEEEK (SEQ ID NO: 46), preferably selected from the group consisting of EEEEEE (SEQ ID NO: 47), EEEEEEEEEE (SEQ ID NO: 11), EEEEEEEEEEEEEEE (SEQ ID NO: 5), EEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 12), EEEEEEEEEEEEEEEEEEEEEEEEE (SEQ ID NO: 48), DDDDDDDDDD (SEQ ID NO: 31), DDDDDDDDDDDDDDD (SEQ ID NO: 32), DDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 33), DDDDDDDDDDDDDDDDDDDDDDDDD (SEQ ID NO: 34), NNNNNNNNNN (SEQ ID NO: 36), NNNNNNNNNNNNNNN (SEQ ID NO: 37), NNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 38), NNNNNNNNNNNNNNNNNNNNNNNNN (SEQ ID NO: 39), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDEDEDEDEDEDEDE (SEQ ID NO: 42), EEEEDEEEED (SEQ ID NO: 13), EEEEDEEEEDEEEED (SEQ ID NO: 14), EEEEDEEEEDEEEEDEEEED (SEQ ID NO: 15), EDDDDEDDDDEDDDD (SEQ ID NO: 41), EEEETEEEET (SEQ ID NO: 19), EEEETEEEETEEEET (SEQ ID NO: 20), and EEEETEEEETEEEETEEEET (SEQ ID NO: 21), ETETETETETETETE (SEQ ID NO: 44), and ETTTTETTTTETTTT (SEQ ID NO: 43).

6. The multifunctional molecule according to any one of the preceding claims, wherein the peptide linker is covalently linked to the N-terminal end of the cytokine and is connecting the antigen binding domain and the cytokine.

7. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises two peptide linkers, a first peptide linker being as described in any one of claims 1-6 and a second peptide linker, wherein:- the second peptide linker has an amino acid sequence as defined in any one of claims 1-7 for the first linker; or- the second peptide linker is identical to the first peptide linker; or- the second peptide linker is between 10 and 30 amino acids in length and consists of less than 50% of basic amino acids and between 50% and 100% of amino acids independently selected from the group consisting of G, E, P, A, V, L, I, M, F, Y, W, Q, N, D, S and T, preferably from the group comprising E, D, P, A, N and T.

8. The multifunctional molecule according to claim 7, wherein:- the first peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the second peptide linker is linked to the C- terminal end of the cytokine; or- the second peptide linker is covalently linked to the N-terminal end of the cytokine and connects the antigen binding domain and the cytokine, and the first peptide linker is linked to the C-terminal end of the cytokine.

9. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises a first peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5) and a second peptide linker comprising or consisting of EEEEEEEEEEEEEEE (SEQ ID NO: 5), preferably wherein:- the N-terminal end of the first peptide linker is covalently linked to the C-terminal end of the antigen binding domain, preferably in C-terminal end of a Fc domain;- the C-terminal end of the first peptide linker is covalently linked to the N-terminal end of the cytokine; and- the N-terminal end of the second peptide linker is covalently linked to the C-terminal end of the cytokine.

10. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises a single antigen binding domain, optionally a Fc domain, and a single cytokine.

11. The multifunctional molecule according to any one of the preceding claims, wherein the multifunctional molecule comprises or consists of: a) a first entity comprising a first Fc chain, said Fc chain being devoid of antigen binding domain and of cytokine; and b) a second entity comprising i) a single antigen binding domain, ii) optionally a peptide spacer, iii) a second Fc chain complementary to the first Fc chain, the first and second Fc chain forming together a Fc domain; and iv) either- the first peptide linker and the single cytokine, variant or fragment thereof; said first peptide linker being covalently linked to the N-terminal or C-terminal end of the cytokine; or- the first and second peptide linkers and the single cytokine, variant or fragment thereof; wherein the first peptide linker is covalently linked to the N-terminal end of the cytokine and the second peptide linker is linked to the C-terminal end of the cytokine.

12. The multifunctional molecule according to any one of the preceding claims, wherein the antiinflammatory cytokine, variant or fragment thereof is a cytokine selected from the group consisting of TGFP, IL-2, IL-4, IL-10, IL-11, IL-12, IL-13, IL-14, IL-19, IL-22, IL-24, IL-25, IL-27, IL-35, IL-37 and IL-38 or a variant thereof having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % of sequence identity with the wildtype cytokine, preferably a cytokine selected from the group consisting of IL-2, IL-10, IL-35, IL-37 and IL-38 or a variant thereof, even more preferably is IL-2 or IL-10 or a variant thereof.

13. The multifunctional molecule according to any one of the preceding claims, wherein the antigen binding domain binds to a target expressed on immune cells surface and being selected from the group consisting of CD3, CD4, CD8, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD119, CD137 / 4- 1BB / TNFRSF9, CD150 / SLAMF1, CD153, CD154 / CD40L, CD28, CD223 / LAG-3, CD226, CD25, CD254, CD26, CD27, CD30, CD39 / ENTPD1, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, LFA-1, TIM-1, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE / CD103, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PD-1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206 / MRC1, LTBR / CD70, TNFSF14, SLAMF7, NKG2A, 2B4, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP,TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR,BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF18 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, I L4R, CLEC-1A, CD21, CLEC- 9A, CD180, CD59, CD54, CD71, CD35, CD74, CD165, 4-1BBL / CD137L, ICOSL / CD275, SIRPa, SIRPG, TREM2, LILRB2, Dectin-1, CLEVER1 and CD160, preferably selected from the group consisting of SIRPa, TREM2, LILRB2, CD206 / MRC1, CD163, CLEC-1A, Dectin-1, CLEVER1 and CLEC-9A.

14. The multifunctional molecule according to any one of the preceding claims , wherein the antigen binding domain is an agonist anti-human SIRPalpha antibody or antigen-binding fragment thereof.

15. The multifunctional molecule according to any one of the preceding claims, wherein the antigen binding domain is a humanized anti-human PD-1 antibody or antigen-binding fragment thereof, preferably comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) selected from the group consisting of: iv) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 74 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 75; v) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 96 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 97; and vi) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 104 and a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 105.

16. The multifunctional molecule according to any one of the preceding claims, wherein the antigen binding domain is an anti-human TIG IT antibody or antigen-binding fragment thereof, preferably comprising or consisting of i) a heavy chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 133 and ii) a light chain variable domain comprising or consisting of an amino acid sequence as set forth is SEQ ID NO: 134.

17. An isolated nucleic acid molecule, a group of isolated nucleic acid molecules or a vector encoding the multifunctional molecule according to any one of claims 1 to 16.

18. A host cell comprising the isolated nucleic acid molecule and / or the group of isolated nucleic acid molecules and / or the vector according to claim 17.

19. A pharmaceutical composition comprising the multifunctional molecule according to any one of claims 1 to 16.

20. The multifunctional molecule according to any one of claims 1-16 or the pharmaceutical composition according to claim 19 for use as a medicament.

21. The multifunctional molecule according to any one of claims 1-16 or the pharmaceutical composition according to claim 19, for use in the treatment of an auto-immune or inflammatory disease or disorder.

22. A method for treating an auto-immune or inflammatory disease or disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the multifunctional molecule according to any one of claims 1-16 or of the pharmaceutical composition of claim 19.

23. Use of a multifunctional molecule according to any one of claims 1-16, in the manufacture of a medicament for treating an auto-immune or inflammatory disease or disorder in a subject in need thereof.

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