Interleukin-2 chimeric constructs with targeting specificity to inflamed tissues

Abstract

The present invention relates to a target-directed chimeric construct comprising i) an interleukin 2 (IL2) moiety and ii) a targeting moiety that binds to an oxidized protein or oxidized lipid. The targeting moiety is preferably an antibody or scFv that binds a specific oxidized protein or oxidized lipid, directing the fusion protein to the target inflamed tissue. The chimeric construct preferably further comprises a beta chain of C4b-binding protein (C4BP) that can form a dimeric protein.

Description

[Technical field]

[0001] The present invention relates to a chimeric construct comprising IL2 and a targeting moiety that confers specificity for inflamed tissue. [Background technology]

[0002] Interleukin-2 (IL2 or IL-2) is a cytokine that regulates key aspects of the immune system. IL2 has been used in attempts to boost immune responses in patients with cancer and autoimmune and / or inflammatory diseases. IL2 is a potent T cell growth factor that promotes immune responses, including clonal expansion of antigen-activated T cells, drives the development of CD4+ T helper (Th)1 [(Th)1] and Th2 cells, terminally differentiates CD8+ cytotoxic T lymphocytes (CTLs), and opposes the development of CD4+ Th17 (Th17) and T follicular helper (Tfh) cells. IL2 also shapes T cell memory recall responses.

[0003] Low doses of IL2 have been used to selectively boost tolerance, thereby suppressing unwanted immune responses associated with autoimmune-like attack of self-tissues. The experience to date is that this therapy is safe and there is no sign of reactivation of autoaggressive T cells, while regulatory T cells (Tregs) are expanded in almost all patients, which is accompanied by clinical improvement.

[0004] Nonetheless, there is room for improvement of IL2 as a therapy, in particular it would be desirable to improve the efficacy of treatment by delivering IL2 to the desired target tissue, thereby increasing its concentration locally in the tissue of interest, and there is also a need to provide IL2 constructs with targeting specificity to limit potential "off-target" effects associated with the ubiquitous presence of lymphocyte populations that can be activated in response to IL-2. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides an IL2 chimeric construct with targeting specificity for inflamed tissues.

[0006] The inventors have surprisingly shown that such target-directed chimeric constructs significantly improve the efficacy of IL2 treatment. In particular, the inventors have shown that the use of the target-directed chimeric constructs of the invention leads to an increase in the number of Tregs recruited to the site of inflammation and to an increase in the proliferation of Tregs. [Means for solving the problem]

[0007] Specifically, the chimeric construct of the present invention comprises: (i) at least one interleukin-2 moiety; and ii) at least one targeting moiety that binds to an oxidized protein or lipid, such as a proinflammatory oxidized protein or lipid.

[0008] In a preferred embodiment, the targeting moiety binds to oxidation-specific epitope (OSE).In a particular embodiment, the targeting moiety binds to (i) malondialdehyde (MDA) epitope, (ii) 2-(ω-carboxyethyl)pyrrole (CEP) epitope, (iii) 4-hydroxynonenal (4-HNE) epitope, or (iv) oxidized phospholipid (OxPL), such as oxidized phospholipid containing phosphocholine (PC-OxPL), oxidized phosphatidylethanolamine (OxPE), oxidized phosphatidylserine (OxPS) or oxidized cardiolipin (OxCL), preferably oxidized phospholipid containing phosphocholine (PC-OxPL).

[0009] In certain embodiments, the targeting moiety is an antibody or an antibody fragment, such as a single chain variable fragment (scFv).

[0010] In certain embodiments, the targeting moiety is selected from the group consisting of an E06 antibody or an E06 antibody fragment such as E06 scFv; an LR04 antibody or an LR04 scFv; an NA17 antibody fragment such as an NA17 antibody or an NA17 scFv; an E014 antibody fragment such as an E014 antibody or an E014 scFv; an MDA2 antibody fragment such as an MDA2 antibody or an MDA2 scFv; an IK17 antibody fragment such as an IK17 antibody or an IK17 scFv; an LR01 antibody fragment such as an LR01 antibody or an LR01 scFv, and functional variants thereof.

[0011] In a preferred embodiment, the targeting moiety is the E06 antibody or an E06 antibody fragment such as the E06 scFv, or a functional variant thereof. In certain embodiments, the E06 scFv comprises: - a variable heavy (VH) domain comprising an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 12; and - a variable light (VL) domain comprising an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 11; Includes.

[0012] In a particular embodiment, the IL-2 moiety is human IL-2 or a homologous variant thereof, which variant has at least 85% amino acid identity with human wild-type IL-2, preferably the variant is an active analogue of human IL-2 having at least 90% amino acid identity with human wild-type IL-2, and preferably said IL-2 moiety is an IL2 mutein comprising a substitution at position N88 of SEQ ID NO:2, more preferably the substitution N88R or N88D.

[0013] In certain embodiments, the IL2 moiety and the targeting moiety are fused in frame or via an amino acid linker, preferably a poly-G linker.

[0014] In a particular embodiment, said chimeric construct further comprises the beta chain of C4b binding protein (C4BPβ) capable of forming a dimeric protein or at least one fragment or functional variant thereof.

[0015] In certain embodiments, the fragment of C4BPβ comprises or consists of amino acid residues 194-252 of C4BPβ, or a longer fragment of C4BPβ extending up to amino acid 135 at the N-terminus.

[0016] In a particular embodiment, the chimeric construct comprises a functional variant of C4BPβ, the functional variant of C4BPβ being a) a modified sequence of a fragment of C4BPβ, in which less than 25 percent, preferably less than 10 percent, of the amino acids of the fragment of C4BPβ have been excised or replaced, and in which the cysteines at positions 202 and 216 and at least three amino acids upstream and downstream of each cysteine ​​are conserved; or b) a modified sequence of a fragment of C4BPβ in which the cysteine ​​responsible for dimerization is replaced by an amino acid, preferably selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine and tryptophan, and another amino acid of the fragment of C4BPβ is replaced by a cysteine; or c) the sequence of a fragment of C4BPβ modified by the insertion of a sequence heterologous to the beta chain between the cysteines responsible for dimerization; or d) The sequence of a fragment of C4BPβ modified by excising the amino acids between the cysteines responsible for dimerization. Includes.

[0017] In a particular embodiment, the IL-2 moiety is fused at the N-terminus of C4BPβ or said fragment thereof, and preferably the C-terminus of C4BPβ or said fragment thereof is fused to the targeting moiety.

[0018] In certain embodiments, the chimeric construct is in a dimeric form, with the monomers associated by a covalent bond between two cysteines of C4BPβ. Homodimers and heterodimers are described in more detail below.

[0019] Another aspect of the invention relates to a nucleic acid encoding the chimeric construct of the invention. The invention also relates to a vector comprising said nucleic acid and to a host cell comprising said nucleic acid or said vector.

[0020] The present invention also relates to the chimeric construct of the invention for use in the treatment of an autoimmune and / or inflammatory disease. [Brief description of the drawings]

[0021] [Figure 1] Figure 1: In vitro evaluation of the functional design of target-directed fusion proteins. Stable HEK 293T cell lines transduced with lentiviral vectors containing transgenes encoding each of the target-directed fusion proteins were cultured for 48 hours in serum-free medium. A) Design of target-directed fusion proteins. B) Detection of target-directed fusion proteins in stable HEK 293T cell supernatants by enzyme-linked immunosorbent assay (ELISA). C) Concentrated and filtered supernatants were then used to characterize IL2-C4bpβ-scFv and IL2N88R-C4bpβ-scFv by Western blot using either a primary polyclonal anti-human IL-2 antibody or a primary monoclonal anti-histidine antibody. D) Functional activity of the target-directed fusion proteins was evaluated by evaluation of STAT5 phosphorylation (pSTAT5) responses in human CD4+ regulatory T cells, CD4+ conventional T cells, CD8+ T cells and NK cells using flow cytometry. [Diagram 2] Figure 2: Therapeutic efficacy of targeted fusion proteins in experimental colitis inflammation models. A) 6-8 week old C57BL / 6(Jrj) female mice (n=6) were injected by intraperitoneal route with AAV 5.1010-5.1011vg (viral genome) encoding IL2, scFvE06, IL2-C4bpβ-scFvE06 or IL2N88R-C4bpβ-scFvE06 or left untreated, and 7 days later, immunized by oral administration of dextran sulfate sodium (2%) diluted in water for 6 days. Immune monitoring analysis of CD4+ Treg and CD25 MFI, CD4+ Tconv CD25+ and NK cells in peripheral blood 7 days after AAV injection was performed (B). Clinical disease assessment was based on weight loss (C), stool form (D), bleeding (E), and disease activity index (F). Immune monitoring analysis of Tregs expressing integrin α4β7 (G) and Ki67 (H) in brachial and para-aortic lymph nodes. Statistical significance was assessed between different treatment groups after calculation of AUC using GraphPad Prism version 6.00 and calculated using the Mann-Whitney test (comparison of means, unpaired test, non-parametric test, two-sided P value), where P<0.05 (*) was interpreted as statistically significant (**P<0.01, ***P<0.001). In all graphs, error bars represent standard error of the mean (SEM). [Diagram 3] FIG. 3 is a graph reporting the pharmacokinetics in mice following a single injection of IL2 or IL2-C4bpβ-scFvE06. [Figure 4] Figure 4 is a group of photographs of immunochemical staining in a DSS-induced colitis mouse model. Only mice treated with IL-2-C4bpβ-scFvE06 showed detection of both IL2 and 6X-HisTag in the colon. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition The "subject" or "patient" to be treated can be any mammal, preferably a human. Human subjects can be pediatric, adult, or geriatric.

[0023] The term "treat" or "treatment" refers to any improvement in a disease. The term includes alleviating at least one symptom or reducing the severity or development of a disease. If the disease is an inflammatory and / or autoimmune disorder, the term more particularly includes reducing the risk, occurrence or severity of acute episodes (flares). The term "treat" or "treatment" includes reducing the progression of a disease. In particular, the present invention includes preventing or slowing down disease progression. The term "treat" or "treatment" further includes prophylactic treatment, especially by reducing risk or delaying the onset of a disease in subjects who are asymptomatic but diagnosed as "at risk".

[0024] "Regulatory T cells" or "Tregs" are T lymphocytes with immunosuppressive activity. Natural Tregs are characterized as CD4+CD25+Foxp3+ cells. Tregs play a major role in controlling inflammatory diseases, but their mode of action in such diseases is not fully understood. Indeed, in most inflammatory diseases, Treg removal exacerbates the disease, whereas Treg addition reduces the disease. Most Tregs are CD4+ cells, but there is also a rare population of CD8+Foxp3+ T lymphocytes with suppressive activity.

[0025] In the context of this application, "effector T cells" (or "Teff") refer to conventional T lymphocytes other than Tregs (sometimes referred to in the literature as Tconv) that express one or more T cell receptors (TCRs) and perform effector functions (e.g., cytotoxic activity, cytokine secretion, etc.). The major population of human Teffs according to the present invention includes CD4+ T helper lymphocytes (e.g., Th0, Th1, Th2, Th9, Th17, Tfh) and CD4+ or CD8+ cytotoxic T lymphocytes, which populations may be specific for self antigens or for non-self antigens. Teffs do not include Foxp3+ regulatory CD8+ T cells.

[0026] In the context of this application, "T follicular helper cells" (or "Tfh") refer to T CD4+ lymphocytes that express BcL6, CXCR5 and PD1, are Foxp3-, and provide B cell help.

[0027] In the context of the present application, "T follicular regulatory cells" (or "Tfr") refer to CD4+CXCR5+PD-1+Bcl6+Foxp3+CD25- T lymphocytes.

[0028] An antibody "specifically binds" to a target antigen if it binds with greater affinity, greater avidity, more readily and / or with greater duration than it binds to other substances. "Specific binding" or "preferential binding" does not necessarily mean (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure.

[0029] An "antibody fragment" includes only a portion of an antibody, where the portion typically retains at least one, and more typically most or all, of the functions normally associated with that portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment contains the antigen-binding site of the original antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, such as one that contains an Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an original antibody, such as FcRn binding, antibody half-life modulation, ADCC function, and complement fixation.

[0030] The antigen-binding region or antigen-binding fragment corresponds to the arms of the Y-shaped structure of an antibody, each of which is composed of an intact light chain paired with the VH and CH1 domains of a heavy chain, and is called a "Fab fragment" (for antigen-binding fragment). Fab fragments were first generated from native immunoglobulin molecules by papain digestion, which cleaves antibody molecules at the hinge region, but on the amino-terminal side of the interchain disulfide bond, thereby releasing two identical antigen-binding arms. Other proteases, such as pepsin, also cleave antibody molecules at the hinge region, but on the carboxy-terminal side of the interchain disulfide bond, thereby releasing a fragment composed of two identical Fab fragments, which remain linked via disulfide bonds; reduction of the disulfide bonds of the F(ab')2 fragment generates a Fab' fragment.

[0031] The part of the antigen-binding region corresponding to the VH and VL domains is called the Fv fragment (for fragment variable); it contains the CDRs (complementarity determining regions) which form the antigen-binding site (also called the paratope).

[0032] The effector region of an antibody, responsible for its binding to effector molecules or cells, corresponds to the stem of the Y-shaped structure, contains the paired CH2 and CH3 domains of the heavy chain (or the CH2, CH3 and CH4 domains, depending on the class of the antibody) and is called the Fc region (for fragment of crystallizable region).

[0033] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. These scFv fragments retain the specificity of the original antibody despite the removal of the constant regions and the introduction of the linker.

[0034] The following sequences are shown in the sequence listing: SEQ ID NO:1 is wild type human IL2 (253 amino acids, including signal peptide) SEQ ID NO:2 is mature wild type human IL2 (233 amino acids, no signal peptide) SEQ ID NO:3 is C4BP beta chain (1-252) SEQ ID NO: 4 is fragment 194-252 of the C4BP beta chain SEQ ID NO:5 is fragment 137-252 of the C4BP beta chain SEQ ID NO:6 is the amino acid sequence of human IL-2 (Hi2cb) fused to the terminal region of C4BPβC, including the signal peptide. SEQ ID NO: 7 is the amino acid sequence of mutant IL-2(N88R) (Hi2mcb) fused to the C-terminal region of C4BPβ, including the signal peptide SEQ ID NO:8 is the GGGGS pattern (linker) SEQ ID NO:9 is the amino acid sequence of human IL-2 (Hi2cb) fused to the C-terminal region of C4BPβ without the signal peptide. SEQ ID NO: 10 is the amino acid sequence of mutant IL-2(N88R)(Hi2mcb) fused to the C-terminal region of C4BPβ without the signal peptide SEQ ID NO: 11 is the sequence of the E06 scFv VL domain SEQ ID NO: 12 is the sequence of the E06 scFv VH domain SEQ ID NO: 13 is the sequence of E06 scFv (without HIS tag) SEQ ID NO: 14 is the linker GGGGSGGGSGGGGGS SEQ ID NO: 15 is the sequence of the target-directed fusion protein IL-2-C4bpβ-scFvE06 (without peptide signal and without HIS tag) SEQ ID NO: 16 is the sequence of the target-directed fusion protein IL-2-C4bpβ-scFvE06 (with peptide signal but without HIS tag) SEQ ID NO: 17 is the sequence of the target-directed fusion protein IL-2N88R-C4bpβ-scFvE06 (without peptide signal and without HIS tag) SEQ ID NO: 18 is the sequence of the target-directed fusion protein IL-2N88R-C4bpβ-scFvE06 (with peptide signal but without HIS tag) SEQ ID NO: 19 is the sequence of the targeting fusion protein IL-2-C4bpβ-scFvE06 (with peptide signal and with HIS tag) SEQ ID NO: 20 is the sequence of the target-directed fusion protein IL-2N88R-C4bpβ-scFvE06 (with peptide signal and with HIS tag)

[0035] The chimeric constructs of the present invention comprise (i) at least one interleukin-2 moiety, and ii) at least one targeting moiety.

[0036] IL-2 part As used herein, interleukin-2 (IL-2) encompasses mammalian wild-type interleukin-2 and variants thereof. Preferably, the IL-2 is human IL-2, or a variant thereof.

[0037] Activated variants of IL-2 have been disclosed in the literature. The variants of native IL-2 can be fragments thereof, analogs thereof and derivatives thereof. By "fragment" is intended a polypeptide comprising only a portion of the polypeptide sequence. "Analog" refers to a polypeptide comprising a native polypeptide sequence with one or more amino acid substitutions, insertions or deletions. Muteins and pseudopeptides are specific examples of analogs. "Derivatives" include any native IL-2 polypeptide or a fragment or analog thereof that has been modified, such as glycosylated, phosphorylated, fused to another polypeptide or molecule, polymerized, etc., or through chemical or enzymatic modification or addition, to improve the properties of IL-2 (e.g., stability, specificity, etc.). The IL-2 portion of the activated variant has, as a whole, at least 75%, preferably at least 80%, 85%, more preferably at least 90%, or at least 95% amino acid sequence identity to the amino acid sequence of a reference IL-2 polypeptide, for example, mature wild-type human IL2.

[0038] As used herein, "wild-type IL-2" means IL-2, whether natural or recombinant, that contains the normally occurring 133 amino acid sequence of native human IL-2, the amino acid sequence of which is set forth in Fujita, et. al., PNAS USA, 80,7437-7441 (1983). SEQ ID NO:2 (133 amino acids) is the human IL-2 sequence minus the signal peptide, which consists of an additional 20 N-terminal amino acids. SEQ ID NO:1 (153 amino acids) is the human IL-2 sequence including the signal peptide.

[0039] As used herein, "IL-2 mutein" refers to a polypeptide in which specific amino acid substitutions are made relative to the human mature interleukin-2 protein. All amino acid numbering is relative to the human mature interleukin-2 protein of SEQ ID NO:2 unless otherwise indicated.

[0040] In some embodiments, the cysteine ​​at position 125 is replaced with a neutral amino acid, such as serine (C125S), alanine (C125A), threonine (C125T) or valine (C125V).

[0041] For example, when the active mutant is expressed in mammalian cells such as CHO or HEK cells, removal of the O-glycosylation site leads to a more homogeneous product.

[0042] In certain embodiments, the activated variant comprises an additional amino acid mutation that removes the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2. In one embodiment, the additional amino acid mutation that removes the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In certain embodiments, the additional amino acid mutation is the amino acid substitution T3A. · Activated IL-2 variants that selectively promote the proliferation, survival, activation and / or function of T-reg cells are particularly useful for the treatment of inflammatory and / or autoimmune disorders.

[0043] By "selectively promoting", it is meant that the active variant promotes activity in T-reg cells but has limited or no ability to promote activity in non-regulatory T cells. Further described herein is an assay to screen for active variants that selectively promote T-reg cell proliferation, survival, activation and / or function. Methods for determining whether a mutant IL-2 polypeptide is active are available in the art. See, for example, WO2016 / 014428. An active variant is defined as a variant that exhibits the ability to stimulate Tregs, including variants with improved, similar or even reduced ability to stimulate Tregs when compared to wild-type IL-2 or aldesleukin (defined below), so long as the variant does not stimulate Teff beyond stimulating Tregs. Methods for testing whether a candidate molecule stimulates T cells, particularly Tregs, or NK cells, are well known. The variants can be tested for their ability to stimulate effector T cells (such as CD8+ T cells), CD4+Foxp3+Tregs, or NK cells. In a preferred embodiment, the active variants show a reduced ability to stimulate NK cells compared to wild-type IL2 or aldesleukin. Monitoring STAT5 phosphorylation is a simple method to evaluate variants for their ability to preferentially stimulate Tregs over Teffs, as described in Yu et al, Diabetes 2015;64:2172-2183. In certain embodiments, variants are particularly useful when a predetermined level of STAT5 phosphorylation is obtained, provided that the dose is 10-fold lower for Tregs than other immune cells, including Teffs.

[0044] The active variant induces signaling events that preferentially induce survival, proliferation, activation and / or function of Treg cells. In certain embodiments, the IL-2 variant retains the ability to stimulate STAT5 phosphorylation and / or phosphorylation of one or more of the downstream signaling molecules of IL-2R, such as p38, ERK, SYK and LCK, in Treg cells. In other embodiments, the IL-2 variant retains the ability to stimulate transcription or protein expression of genes or proteins, such as FOXP3, Bcl-2, CD25 or IL-10, in T-reg cells that are important for survival, proliferation, activation and / or function of T-reg cells. In other embodiments, the IL-2 variant exhibits a reduced ability to stimulate endocytosis of the IL-2 / IL-2R complex on the surface of CD25+ T cells. In other embodiments, the IL-2 variants demonstrate inefficient, reduced or absent stimulation of PI3-kinase signaling, e.g., inefficient, reduced or absent phosphorylation of AKT and / or mTOR (mammalian target of rapamycin). In yet other embodiments, the IL-2 variants retain the ability of wild-type IL-2 to stimulate STAT5 phosphorylation and / or phosphorylation of one or more of the downstream signaling molecules of IL-2R in T-reg cells, and further demonstrate inefficient, reduced or absent phosphorylation of STAT5, AKT, and / or mTOR or other downstream signaling molecules of IL-2R in FOXP3-CD4+ or FOXP3-CD8+ T cells or NK cells. In other embodiments, the IL-2 variants are inefficient or unable to stimulate survival, growth, activation and / or function of FOXP3-CD4+ or FOXP3-CD8+ T cells or NK cells.

[0045] In either case, these variants have the ability to stimulate cell lines such as CTLL-2 or HT-2, which can be widely used to determine their biological activity. By way of example, the biological activity of IL-2 can be determined by a cell-based assay carried out on the HT-2 cell line (clone A5E, ATCC® CRL-1841™), whose proliferation depends on IL-2. The cell proliferation in the presence of a wide variety of test interleukin 2 products is compared with the proliferation recorded with the IL-2 international standard [Second WHO INTERLEUKIN 2 International Standard (human, rDNA derived) NIBSC code: 86 / 500]. [Cell proliferation is measured after the addition of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (inner salt, MTS) and its conversion to formazan by viable and active cells. The formazan concentration is then measured spectrophotometrically at 490 nm.

[0046] Examples of IL-2 variants are disclosed in, for example, EP109748, EP136489, US4,752,585; EP200280, EP118617, WO99 / 60128, EP2288372, US9,616,105, US9,580,486, WO2010 / 085495, WO2016 / 164937.

[0047] By way of example, certain mutations may result in reduced affinity for the signaling chain of the IL-2 receptor (IL-2Rβ / CD122 and / or IL-2Rγ / CD132) and / or reduced ability to induce signaling events from one or both subunits of the IL-2 receptor. Other mutations may confer higher affinity for CD25 (IL-2Rα). In either case, these mutations define active variants that preferentially induce Treg survival, proliferation, activation and / or function. This property can be monitored using surface plasmon resonance.

[0048] Particular examples of useful variants include IL-2 muteins that exhibit at least one amino acid substitution at each of positions D20, N30, Y31, K35, V69, Q74, N88, V91 or Q126, numbered according to wild-type IL-2, meaning that the selected amino acid is identified by reference to the position at which that amino acid normally occurs in the mature sequence of wild-type IL-2 of SEQ ID NO:2.

[0049] Preferred IL-2 muteins contain at least one substitution at each of positions D20H, D20I, D20Y, N30S, Y31H, K35R, V69AP, Q74, N88R, N88D, N88G, N88I, V91K, or Q126L.

[0050] In some embodiments, the IL-2 mutein molecule comprises a V91K substitution. In some embodiments, the IL-2 mutein molecule comprises a N88D substitution. In some embodiments, the IL-2 mutein molecule comprises a N88R substitution. In some embodiments, the IL-2 mutein molecule comprises an H16E, D84K, V91N, N88D, V91K, or V91R substitution, any combination thereof. In some embodiments, these IL-2 mutein molecules also comprise a substitution at position 125 as described herein. In some embodiments, the IL-2 mutein molecule is: T3N, T3A, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20H, D20I, D20Y, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81 and Q126.In some embodiments, the amino acid sequence of the IL-2 mutein molecule differs from the amino acid sequence set forth in the mature IL-2 sequence and has a C125A or C125S substitution as well as any of the following: T3N, T3A, L12G, L12K, L12Q L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81 and R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, Q126I, Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule differs from the amino acid sequence set forth in the mature IL-2 sequence by having a C125A or C125S substitution and one substitution selected from D20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S. In some embodiments, the IL-2 mutein comprises an N88R and / or D20H mutation.

[0051] These substitutions may be used alone or in combination with each other. In some embodiments, the mutein contains each of these substitutions. In some embodiments, the mutein contains 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations.

[0052] In some embodiments, the IL-2 mutein comprises an N88R or N88D mutation, preferably N88R. In some embodiments, the IL-2 mutein comprises a C125A or C125S mutation. These substitutions may be used alone or in combination with each other. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises each of these substitutions.

[0053] In a particular embodiment, the IL-2 moiety is aldesleukin. Aldesleukin is the active ingredient of Proleukin®. Aldesleukin is a variant of mature human IL-2 that contains two amino acid modifications compared to the sequence of mature human IL-2 (SEQ ID NO:2): deletion of the first amino acid (alanine) and substitution of cysteine ​​at position 125 with serine. Conservative modifications and substitutions at other positions of IL-2 (i.e., those that have minimal effect on the secondary or tertiary structure of the mutein) are included. Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and by Argos in EMBO J., 8: 779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: -ala, pro, gly, gln, asn, ser, thr; -cys, ser, tyr, thr; -val, ile, leu, met, ala, phe; -lys, arg, his; -phe, tyr, trp, his; and -asp, glu.

[0054] Variants with mutations that disrupt binding to the α subunit of IL-2R are not preferred, as these mutant forms may have a reduced ability to stimulate Tregs. · Activated IL-2 variants that promote proliferation, survival, activation and / or function of Teff cells may be useful in treating cancer.

[0055] Such activated mutants of IL-2 each contain at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor (CD25) and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, compared to the wild-type IL-2 polypeptide, a property that can be monitored using surface plasmon resonance.

[0056] Preferred active mutants include IL-2 muteins containing the following substitutions: F42A, K43N, Y45A, and / or E62A.

[0057] Activated variants, such as mutant forms of human IL-2 (hIL-2) with reduced affinity for CD25, can be generated, for example, by amino acid substitutions at amino acid positions 35, 38, 42, 43, 45, 62, or 72, or combinations thereof (numbered relative to human IL-2 SEQ ID NO:2). Exemplary amino acid substitutions include 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, and combinations thereof. , Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, E62G, E62A, E62S, E62T, E62Q, E62E, E62N, E62D, E62R, E62K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Particular active mutants useful in the chimeric constructs of the invention contain amino acid mutations at amino acid positions corresponding to residues 42, 45 or 72 of human IL-2, or combinations thereof. In one embodiment, said amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, more particularly an amino acid substitution selected from the group of F42A, Y45A and L72G. These activated mutants exhibit substantially similar binding affinity to the intermediate affinity IL-2 receptor and have substantially reduced affinity to the α subunit of the IL-2 receptor and the high affinity IL-2 receptor (IL2Rαβγ) compared to the wild-type IL-2 mutant.

[0058] Other characteristics of useful active variants include: the ability to induce proliferation of IL-2 receptor-bearing T cells and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T cells and / or NK cells, the ability to produce interferon (IFN)-y as a secondary cytokine by NK cells, a reduced ability to induce synthesis of secondary cytokines, particularly IL-10 and TNF-a, by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.

[0059] A particular activated variant contains three amino acid mutations that eliminate or reduce the affinity of the activated variant for the α-subunit of the IL-2 receptor, but preserve the affinity of the activated variant for the intermediate affinity IL-2 receptor. In one embodiment, the three amino acid mutations are at positions corresponding to residues 42, 45 and 72 of human IL-2. In one embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In a particular embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 2).

[0060] In a particular embodiment, the amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the α subunit of the IL-2 receptor by at least 5-fold, particularly at least 10-fold, more particularly at least 25-fold. In embodiments where there are two or more amino acid mutations that reduce the affinity of the activated variant to the α subunit of the IL-2 receptor, the combination of these amino acid mutations can reduce the affinity of the activated variant to the α subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment, the amino acid mutation or combination of amino acid mutations abolishes the affinity of the activated variant to the α subunit of the IL-2 receptor, such that binding is undetectable by surface plasmon resonance.

[0061] Substantially similar binding to the intermediate affinity receptor, i.e., maintenance of the affinity of the mutant IL-2 polypeptide for said receptor, is obtained when the activated variant exhibits more than about 70 percent of the affinity possessed by the wild type of the IL-2 mutant for the intermediate affinity IL-2 receptor. Activated variants useful in the present invention can exhibit more than about 80 percent, or even more than about 90 percent, of such affinity.

[0062] The reduced affinity of IL-2 for the α-subunit of the IL-2 receptor combined with the elimination of O-glycosylation of IL-2 results in an IL-2 protein with improved properties. In certain embodiments, the activated variants are capable of eliciting one or more cellular responses selected from the group consisting of: proliferation in activated T lymphocyte cells, differentiation in activated T lymphocyte cells, cytotoxic T cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in natural killer (NK) cells, cytotoxic activity in NK cells, differentiation in NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) antitumor cytotoxicity.

[0063] In some embodiments, these active variants also contain a substitution at position 125 as described herein.

[0064] Targeting section The chimeric construct further comprises a targeting moiety capable of directing IL-2 to targeted inflamed tissue.

[0065] In particular, the targeting moiety binds to oxidized protein or oxidized lipid.In the context of the present invention, the oxidized protein or oxidized lipid is found in inflamed tissue.In certain embodiments, the targeting moiety binds to oxidized protein or oxidized lipid that contributes to inflammation or is involved in inflammatory response.In certain embodiments, the targeting moiety binds to oxidized protein or oxidized lipid that is induced by and / or contributes to oxidative damage and inflammation.

[0066] In certain embodiments, the targeting moiety binds to proinflammatory oxidized proteins or oxidized lipids. "Proinflammatory" refers to oxidized proteins or oxidized lipids that are active mediators of inflammation. For example, proinflammatory proteins or lipids can induce the secretion of inflammatory cytokines and / or the recruitment of effector cells, such as monocytes or macrophages. Proinflammatory cytokines are a type of signaling molecule secreted by immune cells, such as helper T cells (Th) and macrophages, as well as certain other cell types that promote inflammation. These include interleukin-1 (IL-1), IL-12, IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), or granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0067] In certain embodiments, oxidized proteins or lipids are involved in or trigger sterile inflammation. "Sterile inflammation" refers to inflammation that occurs in the absence of any microorganisms and is triggered in response to damage-associated molecular patterns (DAMPs) that are locally released in response to tissue injury. DAMPs are intracellular and extracellular host-derived molecules that are not normally sensed by the immune system, but which become released or modified into denatured self-molecules upon tissue injury. Similar to pathogen-induced inflammation, sterile inflammation is triggered by activation of the innate immune response through recognition of DAMPs by pattern recognition receptors (PRRs), resulting in increased secretion of cytokines and chemokines. Membrane-bound PRRs, such as Toll-like receptors (TLRs), and intracellular PRRs, such as inflammasomes, are key mediators of sterile inflammation. Cytokines belonging to the interleukin-1 (IL-1) family have been proposed to be important drivers of sterile inflammation. Increased secretion of cytokines and chemokines at the site of initial injury ultimately leads to enhanced recruitment of immune cells such as neutrophils and macrophages. Resolution of sterile inflammation should result in tissue repair and re-establishment of homeostasis. Unresolved sterile inflammation has been implicated in the development of several pathologies, including autoimmune diseases, gout, Alzheimer's disease, and atherosclerosis.

[0068] In certain embodiments, the targeting moiety binds to an "oxidation-specific epitope" (OSE), which is present on an oxidized protein or an oxidized lipid, as defined above.

[0069] Cells generate reactive oxygen species (ROS), the biological effects of which depend on the amount produced. At low concentrations, ROS are involved in proliferation, differentiation and cell metabolism, whereas at high concentrations, they are involved in the formation of neutrophil extracellular traps (NETs) that facilitate microbial clearance. In response to certain exogenous and endogenous stimuli, such as inflammation, this balance can be disrupted, which can lead to the accumulation of ROS involved in oxidative stress that causes irreversible changes in DNA, RNA, proteins and lipids. The main consequence of oxidative stress is lipid peroxidation, which generates several highly reactive degradation products that react with lipids, apoproteins and proteins, thereby forming stable covalent adducts and creating "oxidation specific epitopes" (OSEs). OSEs can be found as isolated lipids or can be covalently associated with proteins. Examples of OSEs include oxidized phospholipids (OxPLs). For reviews and examples, see, e.g., Binder et al, (2016) Nat Rev Immunol,16(8):485-97. OSEs can be generated by modification of proteins with truncated phospholipids such as oxidized phosphatidylcholine, oxidized cardiolipin (OxCL), oxidized phosphatidylserine (OxPS) and oxidized phosphatidylethanolamine (OxPE). Other examples of OSEs include the malondialdehyde (MDA) epitope, the 2-(ω-carboxyethyl)pyrrole (CEP) epitope and the 4-hydroxynonenal (4-HNE) epitope.

[0070] OSEs have been documented in oxidized lipoproteins and on the surface of dying cells and circulating microparticles, demonstrating their ability to trigger robust proinflammatory responses [Tsiantoulas et al. (2015). Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies1. J. Lipid Res. 56, 440-448]. In particular, recognition of OSEs, which are categorized as PAMPs ("pathogen-associated molecular patterns"), by PRRs ("pattern recognition receptors") induces the expression of proinflammatory cytokines and activation of cellular effectors such as monocytes and macrophages [Miller et al. (2011). Oxidation-Specific Epitopes are Danger Associated Molecular Patterns Recognized by Pattern Recognition Receptors of Innate Immunity. Circ. Res. 108, 235-248.].

[0071] Since OSE-modified proteins or lipids accumulate under inflammatory conditions, these can be used as targets to direct IL-2 moieties to targeted inflamed tissues.

[0072] In certain embodiments, the targeting moiety binds to an OSE, where the OSE is: a malondialdehyde (MDA) epitope; a 2-(ω-carboxyethyl)pyrrole (CEP) epitope; a 4-hydroxynonenal (4-HNE) epitope, an oxidized phospholipid (OxPL), a phosphocholine-containing oxidized phospholipid (PC-OxPL), an oxidized phosphatidylethanolamine (OxPE), an oxidized phosphatidylserine (OxPS), or an oxidized cardiolipin (OxCL).

[0073] In a preferred embodiment, the targeting moiety binds to an oxidized phospholipid (OxPL).

[0074] Preferably, the targeting moiety binds to phosphocholine-containing oxidized phospholipids (PC-OxPL).The oxidized phospholipids containing phosphocholine head group have been shown to be highly proinflammatory and proatherogenic, and are induced by and propagate oxidative damage and inflammation.The oxidized phospholipids are present in a wide range of inflammatory diseases, including atherosclerosis, rheumatoid arthritis, diabetic nephropathy, multiple sclerosis, and other CNS diseases, as well as a range of acute and chronic lung diseases.

[0075] In certain embodiments, the targeting moiety is an antibody or an antibody fragment, such as a Fab, Fab', F(ab')2, Fv or scFv fragment.

[0076] In a preferred embodiment, the targeting moiety is an scFv fragment, which in fact lacks the Fc domain and therefore has silent effector functions.

[0077] In certain embodiments, the targeting moiety is selected from the group consisting of: an E06 antibody or an E06 antibody fragment such as E06 scFv; an LR04 antibody or an LR04 scFv; an NA17 antibody fragment such as an NA17 antibody or an NA17 scFv; an E014 antibody fragment such as an E014 antibody or an E014 scFv; an MDA2 antibody fragment such as an MDA2 antibody or an MDA2 scFv; an IK17 antibody fragment such as an IK17 antibody or an IK17 scFv; or an LR01 antibody fragment such as an LR01 antibody or an LR01 scFv.

[0078] In a preferred embodiment, the targeting moiety is an E06 antibody or an E06 antibody fragment, such as the Fab, Fab', F(ab')2, Fv or scFv fragment of the E06 antibody, or a functional variant thereof. E06 is a natural IgM autoantibody cloned from apolipoprotein E-deficient mice (apoE- / -), which binds to the phosphocholine (PC) head group of oxidized phospholipids but not to the phosphocholine (PC) head group of normal phospholipids [Friedman et al. (2001) Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids: Importance of Schiff base formation and Aldol condensation. J Biol Chem.;277:7010-7020]. Interestingly, E06 is structurally and functionally identical to the classical "natural" mouse T15 anti-PC antibody, which is reported to be of B1 cell origin and to provide optimal protection from pathogenic pneumococcal infection.

[0079] In certain embodiments, the targeting moiety is E06 scFv or a functional variant thereof.

[0080] The term "functional variant" or "derivative" refers to a sequence that differs from the parent sequence to which it refers by the deletion, substitution or insertion of one or several amino acids without substantially affecting the function of the antibody or its fragment. Preferably, the functional variant exhibits 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity with the native sequence. A functional variant of an antibody or its fragment retains a similar antigen binding affinity compared to the reference antibody (e.g., has a KD of less than 1×10-7M, 10-8M, preferably less than 1×10-9M or less than 1×10-10M). The affinity of binding is defined by the terms ka (association rate constant), kd (dissociation rate constant) or KD (equilibrium dissociation). Typically, when used with respect to antibodies, specifically binding refers to an antibody that specifically binds to ("recognizes") its target with an affinity (KD) value of less than 10-7 M, preferably less than 10-8 M, such as less than 10-9 M or less than 10-10 M. A lower KD value represents a higher binding affinity (i.e., stronger binding), such that a KD value of 10-9 indicates a higher binding affinity than a KD value of 10-8.

[0081] In a specific embodiment, the E06 scFv is: - a variable heavy (VH) domain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 12; and - a variable light chain (VL) domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 11; Includes.

[0082] In a particular embodiment, the VH and VL domains of E06 scFv are fused via an amino acid linker. The term "linker" refers to a (poly)peptide comprising 5-80 amino acids, preferably 5-30, more preferably 10-20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises a GGGGS (SEQ ID NO: 8) repeat sequence. Linkers composed of small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility and allow mobility of the functional domains they connect. In a preferred embodiment, the linker is a linker of SEQ ID NO: 14 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 14.

[0083] In certain embodiments, the E06 scFv fragment comprises or consists of the amino acid sequence set forth in SEQ ID NO:13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO:13.

[0084] In another specific embodiment, the targeting moiety is an LR04 antibody or an LR04 antibody fragment, such as an Fab, Fab', F(ab')2, Fv or scFv fragment of the LR04 antibody, or a functional variant thereof. LR04 is a monoclonal IgM antibody against the MDA epitope cloned from mouse Ldlr- / - spleens undergoing an atherogenic diet (Amir et al. Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease. J Lipid Res. 2012;53:1316-1326).

[0085] In another specific embodiment, the targeting moiety is an NA17 antibody or an NA17 antibody fragment, such as the Fab, Fab', F(ab')2, Fv or scFv fragment of the NA17 antibody, or a functional variant thereof. NA17 is an MDA-specific natural mAb cloned from the spleen of B1 cell-reconstituted Rag1- / - mice [Chou et al. Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans. J Clin Invest. 2009;119(5):1335-1349].

[0086] In another specific embodiment, the targeting moiety is an E014 antibody or an E014 antibody fragment, such as the Fab, Fab', F(ab')2, Fv or scFv fragment of the E014 antibody, or a functional variant thereof. E014 is a monoclonal IgM NAb cloned from the spleen of atherosclerotic Apoe- / - mice, which has been shown to bind to the MDA epitope [Palinski et al. (1996). Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma. J. Clin. Invest. 98, 800-814].

[0087] In another specific embodiment, the targeting moiety is MDA2 antibody or MDA2 antibody fragment, such as Fab, Fab', F(ab')2, Fv or scFv fragment of MDA2 antibody, or functional variants thereof. MDA2 is a mouse monoclonal IgG type antibody specific to MDA-lysine epitope. MDA2 specifically binds MDA-LDL and other MDA modified proteins [Rosenfeld et al (1990). Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis.;10:336-349].

[0088] In another specific embodiment, the targeting moiety is an IK17 antibody or an IK17 antibody fragment, such as an Fab, Fab', F(ab')2, Fv or scFv fragment of the IK17 antibody, or a functional variant thereof. IK17 is a human monoclonal IgG antibody fragment that binds to MDA-LDL and copper OxLDL [Shaw et al. (2001) Human-derived anti-oxidized LDL autoantibody blocks uptake of oxidized LDL by macrophages and localizes to atherosclerotic lesions in vivo. Arterioscler Thromb Vasc Biol.;21:1333-1339]. IK17 was isolated from a phage display library from patients with coronary artery disease who had high plasma autoantibody titers against MDA-LDL. As a human autoantibody, IK17 has potential advantages over murine antibodies, including improved pharmacokinetics and reduced immune responses.

[0089] In another specific embodiment, the targeting moiety is an LRO1 antibody or an LRO1 antibody fragment, such as the Fab, Fab', F(ab')2, Fv or scFv fragment of the LRO1 antibody, or a functional variant thereof. LRO1 is a germline-encoded NAb isolated from the spleen of atherosclerotic Ldlr- / - mice. It was found that LRO1 is directed against oxidized cardiolipin, but not against native cardiolipin [Tuominen et al. (2006). A natural antibody to oxidized cardiolipin binds to oxidized low-density lipoprotein, apoptotic cells, and atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 26, 2096-210].

[0090] Any moiety having dimerization properties A chimeric construct of the invention comprising an IL-2 moiety and a targeting moiety may suitably comprise a moiety that has multimerizing properties, ie a fragment or moiety capable of forming a multimeric protein.

[0091] Preferably, the chimeric construct of the invention comprising an IL-2 moiety and a targeting moiety may suitably comprise a moiety having dimerisation properties, ie a fragment or moiety capable of forming a dimeric protein.

[0092] For example, the chimeric construct can further comprise an Fc fragment of IgG or a functional variant thereof, which has the ability to form at least one dimer, such as a homodimer or heterodimer, trimer, tetramer or any multimer containing a different number of chimeric constructs.

[0093] In certain embodiments, the chimeric construct of the present invention comprising an IL-2 portion and a targeting portion may optionally comprise the beta chain of C4b binding protein (C4BPβ), or at least one fragment thereof or a functional variant thereof, capable of forming a dimeric protein.

[0094] C4BPβ or C4BPβ fragment C4BP protein is involved in the coagulation and complement systems. The major form of C4BP is composed of seven identical 75 kD alpha chains and one 45 kD beta chain. The alpha and beta chains contain eight and three SCR (short consensus repeat) domains, respectively, and these motifs are found in many complement control proteins and consist of 50-70 amino acids organized into beta sheets. The amino acid sequence of the beta chain of human C4BP is shown as SEQ ID NO:3.

[0095] The nucleic acid sequence corresponding to this polypeptide sequence has also been described by Hillarp and Dahlback (1990, PNAS, vol 87, pp 1183-1187). The role of the alpha chain in polymerizing the C4BP protein has been investigated by Kask et al (Biochemistry 2002, 41, 9349-9357). These authors showed that the C-terminal part of the alpha chain, in particular its helical structure and the presence of two cysteines, is necessary for the polymerization of the C4BP protein when the alpha chain is expressed in a heterologous system.

[0096] European Patent Application No. 2227030 describes the production of heteromultimeric recombinant proteins by using C-terminal fragments of the alpha and beta chains of the C4BP protein in fusion with a polypeptide of interest. US Patent No. 7,884,190 describes the use of the beta chain of the C4BP protein in conjunction with the alpha chain of the C4BP protein to produce a dimeric protein, without relying on the use of the beta chain of the C4BP protein in combination with the alpha chain of the C4BP protein.

[0097] The C4BP protein used to carry out the invention is advantageously a human C4BP protein. In a preferred embodiment, the chimeric construct comprises a fragment of the C4BP β chain comprising or consisting of at least amino acids 194 to 252 (SEQ ID NO: 4).

[0098] Sequences encoding longer fragments of the beta chain, or even the entire beta chain, can also be used. In certain applications, it is preferable to avoid using sequences encoding beta chains capable of binding the S protein involved in coagulation. If the selected sequence encodes a fragment containing the first two SCR motifs of the beta chain, these will preferably be versions mutated by addition, deletion or substitution of amino acids to cut out the possibility of interaction with the S protein. SCR motifs and / or [GS] domains can be added to modify, for example increase, the flexibility of the resulting fusion polypeptide or to allow the chimeric protein to adopt a conformation suitable for forming multimers, in particular dimers.

[0099] Longer fragments of C4BPβ can be used, extending up to amino acid 135 at the N-terminus.

[0100] In certain embodiments, a fragment of the C4BP β chain can comprise or consist of at least amino acids 185-252, 180-252, 175-252, 170-252, 165-252, 160-252, 155-252, 150-252, 145-252, 140-252, or 135-252 (relative to SEQ ID NO: 3).

[0101] In a particular embodiment, the fragment of the C4BP β chain comprises or consists of at least amino acids 137 to 252 (SEQ ID NO:5).

[0102] Functional variants of C4BPβ can be used that maintain the ability to form at least one dimer, such as a homodimer or heterodimer, trimer, tetramer or any multimer containing a different number of chimeric constructs.

[0103] Within the context of the present invention, the term "functional variant of a fragment of the C4BP beta chain" refers to a polypeptide sequence modified with respect to the sequence of the fragment of the beta chain by deletion, substitution or addition of one or more amino acids, but said modified sequence retains the ability to form at least a dimeric protein using the method of the present invention. More precisely, the production of a dimeric protein using a sequence encoding a functional variant of the fragment can be at least 80%, preferably at least 90%, even more preferably 95% equivalent to that obtained with the native sequence encoding the fragment (SEQ ID NO: 3 or a fragment thereof) in the same expression system. Preferably, the variant is such that more than 80% of the fusion polypeptides it contains are produced in dimeric form in a eukaryotic expression system according to the present invention.

[0104] In a particular embodiment, the mutant of the beta chain fragment is encoded by a nucleic acid capable of hybridizing under stringent conditions with the wild-type sequence encoding the fragment, as described by Hillarp and Dahlback (1990, PNAS, Vol. 87, pp 1183-1187). The term "stringent conditions" refers to conditions that allow specific hybridization of two single-stranded DNA sequences at about 65°C, for example in a solution of 6*SSC, 0.5% SDS, 5*Denhardt's solution and 100 μg of non-specific DNA or any other solution with equivalent ionic strength, and after washing at 65°C, for example in a solution of up to 0.2*SSC and 0.1% SDS or any other solution with equivalent ionic strength.

[0105] Preferably, the nucleotide sequence encoding a functional variant of the wild-type fragment and hybridizing under stringent conditions with the sequence encoding the fragment has a length in its hybridization portion that is at least 50%, preferably at least 80%, of the length of the sequence encoding the fragment. In a particular implementation, the nucleotide sequence encoding a functional variant of the fragment and hybridizing under stringent conditions with the sequence encoding the fragment has a length in its hybridization portion that is substantially the same as the length of the sequence encoding the fragment.

[0106] In a further implementation, the functional variant is a modified sequence of the wild-type fragment in which one or more amino acids not essential for the dimerization function have been removed or replaced and / or one or more amino acids essential for dimerization have been replaced by amino acids with equivalent functional groups (conservative substitutions). It is particularly recommended to preserve the two cysteines at positions 201 and 215 and the peptide structure around these cysteines to allow the formation of disulfide bridges necessary for dimerization, for example by preserving at least three amino acids upstream and downstream of each cysteine. In particular, functional variants can also be obtained by inserting a heterologous sequence of the beta chain, in particular the domain of the alpha chain of C4BP, between the cysteines responsible for dimerization, or, in contrast, by eliminating certain amino acids present between the same cysteines in question. Alternatively, functional variants can be generated by point modifications of certain amino acids, in particular by substitution of cysteines responsible for dimerization with neutral amino acids with respect to their participation in the dimerization process (e.g., amino acids A, V, F, P, M, I, L and W), and by substitution of other amino acids with cysteines, so as to simultaneously preserve the ability to form intrachain and / or interchain disulfide bridges between cysteines. These modifications thus result in a change in the distance between the various cysteines involved in the multimerization process, in particular dimerization.

[0107] Preferably, less than 50% of the amino acids of the 194-252 fragment are removed or replaced, preferably less than 25% or even less than 10% (e.g. 5 or fewer amino acids) or less than 5% (e.g. 1 or 2 amino acids).

[0108] In certain embodiments, the functional variant comprises or consists of: a) a modified sequence of a fragment of C4BPβ (preferably the 194-252 fragment), in which less than 25 percent, preferably less than 10 percent, of the amino acids of the fragment of C4BPβ (preferably the 194-252 fragment) have been truncated or replaced, and in which the cysteines at positions 202 and 216 (numbered relative to SEQ ID NO: 3) and at least three amino acids upstream and downstream of each cysteine ​​are conserved; or b) a modified sequence of a fragment of C4BPβ (preferably the fragment 194-252), in which the cysteine ​​responsible for dimerization is replaced by an amino acid, preferably selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine and tryptophan, and another amino acid of the fragment of C4BPβ is replaced by a cysteine; or c) the sequence of a fragment of C4BPβ (preferably the fragment 194-252) modified by the insertion of a sequence heterologous to the beta chain between the cysteines responsible for dimerization; or d) The sequence of a fragment of C4BPβ (preferably the fragment 194-252) modified by truncating the amino acids between the cysteines responsible for dimerization.

[0109] Chimeric constructs Preferably, the IL2 moiety and the targeting moiety of the chimeric construct are fused to each other. The IL2 moiety may be fused to the N-terminus or C-terminus of the targeting moiety. In a preferred embodiment, the IL-2 moiety is fused to the N-terminus of the targeting moiety. Preferably, the C-terminus of the IL-2 moiety is fused to the N-terminus of the targeting moiety.

[0110] The IL2 moiety and the targeting moiety are fused in frame (directly) or via an amino acid linker, preferably a poly-G linker. In the context of the present invention, the term "linker" refers to a (poly)peptide comprising 5 to 80 amino acids, preferably 5 to 30, more preferably 10 to 20 amino acids. Suitable linkers are known in the art. In some embodiments, the linker comprises a GGGGS (SEQ ID NO: 8) repeat sequence, but the skilled artisan will understand that other sequences can also be used according to general recommendations [Argos, 1990, J Mol Biol. 20;211(4):943-58; George R, Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. 2002;15:871-879]. Linkers composed of small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids provide flexibility and allow mobility of the functional domains they connect. In a preferred embodiment, the IL2 moiety and the targeting moiety are linked via a linker of SEQ ID NO:14 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO:14.

[0111] In certain embodiments, the chimeric construct further comprises a moiety with dimerization properties, such as C4BPβ or a functional fragment thereof, as described above. Such chimeric constructs preferably form homodimers, but may alternatively be used to generate heterodimers, as described below. In certain embodiments, (i) the IL2 moiety, (ii) the targeting moiety, and (iii) the moiety with dimerization properties are fused to each other in frame (directly) or via an amino acid linker.

[0112] In a preferred embodiment, the IL-2 moiety is fused at the N-terminus of C4BPβ or said functional fragment thereof. Preferably, the IL-2 moiety is fused at the N-terminus of C4BPβ or said functional fragment thereof via an amino acid linker, preferably a poly-G linker. In some embodiments, the IL-2 moiety is fused to the C4BPβ moiety via a linker comprising a GGGGS (SEQ ID NO:8) repeat sequence, preferably a linker of SEQ ID NO:14 or a linker having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO:14.

[0113] In a specific embodiment, the amino acid sequence corresponding to the IL-2 portion fused to the C4BPβ portion (“IL2-C4BPβ”) comprises or consists of SEQ ID NO:9 or SEQ ID NO:10.

[0114] In a preferred embodiment, the C-terminus of the IL-2 moiety is fused to the N-terminus of C4BPβ or said functional fragment thereof, in which the C-terminus of C4BPβ or said fragment thereof is linked to the N-terminus of the targeting moiety.

[0115] In certain embodiments, the chimeric construct comprises, from the N-terminus to the C-terminus: - an IL-2 moiety; - a linker if appropriate, - Targeting part and Includes.

[0116] In a preferred embodiment, the chimeric construct comprises, from the N-terminus to the C-terminus: - an IL-2 moiety; - a linker if appropriate, - a moiety having dimerization properties, such as C4BPβ or a functional fragment thereof; - a linker if appropriate, - Targeting part and Includes.

[0117] In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO: 15 to 20, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 15 to 20. In a preferred embodiment, the chimeric construct comprises or consists of the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 17, or comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to SEQ ID NO: 15 or SEQ ID NO: 17.

[0118] In another specific embodiment, the chimeric construct comprises, from the N-terminus to the C-terminus: - The targeting part, - a linker if appropriate, - IL-2 part and Includes.

[0119] In a preferred embodiment, the chimeric construct comprises, from the N-terminus to the C-terminus: - The targeting part, - a linker if appropriate, - a moiety having dimerization properties, such as C4BPβ or a functional fragment thereof; - a linker if appropriate, - IL-2 part and Includes.

[0120] Homodimeric and heterodimeric constructs Provided herein is a method for producing a recombinant dimeric protein, comprising: a) treating a host cell with i) at least one interleukin 2 (IL2) moiety, ii) a portion capable of forming a dimeric protein, such as the beta chain of C4b binding protein (C4BPβ), or at least one fragment or functional variant thereof, and iii) transfecting with a vector allowing the expression of a nucleotide sequence encoding a chimeric construct, which is a fusion polypeptide comprising at least one targeting moiety as described above; b) culturing the transfected cells under conditions suitable for expressing the nucleotide sequence encoding the fusion polypeptide and the covalent association of two fusion polypeptides in vivo to form a dimeric protein; c) recovering, and preferably purifying, the dimeric protein formed; A method is described that includes:

[0121] Preferably, the transfected cells do not contain any nucleic acid allowing the expression of a nucleotide sequence encoding the C-terminal fragment of the alpha chain of the C4BP protein which is involved in the polymerization of the C4BP protein.

[0122] In certain embodiments, a method of generating a heterodimer is described herein, the method comprising: a. Transfecting a host cell with one or more vectors: i. a first fusion polypeptide comprising i) at least one interleukin 2 (IL2) moiety, and ii) a portion capable of forming a dimeric protein, such as the beta chain of C4b binding protein (C4BPβ), or at least one fragment or functional variant thereof; ii. a second fusion polypeptide comprising i) at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof, and ii) a portion capable of forming a dimeric protein, such as the beta chain of C4b-binding protein (C4BPβ), or at least one fragment or functional variant thereof; 2. enabling expression of one or more nucleotide sequences encoding: b. culturing the transfected cells under conditions suitable for expressing the sequence or sequences of nucleotides encoding the first and second fusion polypeptides and the association of the two fusion polypeptides in vivo to form a heterodimeric protein; c. Recovering, and preferably purifying, the formed heterodimeric protein; Includes.

[0123] In certain embodiments, a method of generating a heterodimer is described herein, the method comprising: a. Transfecting a host cell with one or more vectors: i. a first fusion polypeptide comprising i) at least one interleukin 2 (IL2) moiety, ii) a moiety capable of forming a dimeric protein, such as the beta chain of C4b binding protein (C4BPβ), or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof; ii. a second fusion polypeptide comprising i) at least one heterologous polypeptide defined as different from the interleukin-2 (portion) of the first fusion polypeptide, ii) a portion capable of forming a dimeric protein, e.g. the beta chain of C4b-binding protein (C4BPβ), or at least one fragment or functional variant thereof, and iii) optionally at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof; enabling expression of one or more nucleotides encoding b. culturing the transfected cells under conditions suitable for expressing the sequence or sequences of nucleotides encoding the first and second fusion polypeptides and the association of the two fusion polypeptides in vivo to form a heterodimeric protein; c. Recovering, and preferably purifying, the formed heterodimeric protein; Includes.

[0124] Preferably, in the second fusion polypeptide, C4BPβ or said fragment is fused to the C-terminus of said heterologous polypeptide.

[0125] The term "different" when referring to a heterologous polypeptide means a polypeptide having a primary amino acid sequence which differs by at least one amino acid from the primary sequence of the interleukin-2 (part) of the first fusion polypeptide. Alternatively, the term "different" also covers heterologous polypeptides having the same primary sequence but with different post-translational modifications, for example in terms of acetylation, amidation, biotinylation, carboxylation, hydroxylation, methylation, phosphorylation or sulfation, or by adding lipids (isoprenylation, palmitoylation and myristoylation), carbohydrates (glycosylation) or polypeptides (ubiquitination).

[0126] In a preferred embodiment, the heterologous polypeptide is not IL2.

[0127] Such heterodimeric proteins are also part of the present invention.

[0128] In a particular embodiment, the host cell allows the co-expression of two fusion polypeptides, namely a first fusion polypeptide A comprising i) at least one interleukin 2 (IL2) moiety, ii) a beta chain of C4b binding protein (C4BPβ) capable of forming a dimeric protein or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof; and a second fusion polypeptide A comprising i) at least one interleukin 2 (IL2) moiety, ii) a beta chain of C4b binding protein (C4BPβ) capable of forming a dimeric protein or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof. In this particular embodiment, the co-expression of two fusion polypeptides may also allow the generation of homodimeric AA.

[0129] In a particular embodiment, the host cell allows the co-expression of two fusion polypeptides, namely a first fusion polypeptide A comprising i) at least one interleukin 2 (IL2) moiety, ii) the beta chain of C4b binding protein (C4BPβ) capable of forming a dimeric protein or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof; and a second fusion polypeptide B comprising i) at least one heterologous polypeptide, ii) the beta chain of C4b binding protein (C4BPβ) capable of forming a dimeric protein or at least one fragment or functional variant thereof, and iii) at least one targeting moiety as described above, such as E06 scFv or a functional variant thereof; wherein said heterologous polypeptide is defined as different from the interleukin 2 (moiety) of the first fusion polypeptide. In this particular embodiment, co-expression of two fusion polypeptides may allow the production of homodimers AA and BB as well as the production of the heterodimer AB.

[0130] Also provided is a recombinant eukaryotic cell that allows the synthesis of a dimeric or heterodimeric protein as defined above and that can be obtained by carrying out step a) of the production method defined above. Details regarding production in a host cell are described below.

[0131] Generation method Chimeric constructs in the form of fusion proteins, as well as homodimers or heterodimers thereof, can be produced by DNA recombinant techniques in suitable expression vectors or by RNA molecules.

[0132] The expression vector is selected according to the host cell that the construct is introduced into.Preferably, the expression vector is selected from vectors that allow expression in eukaryotic cells, in particular from chromosomal vectors or episomal vectors or virus derivatives, in particular from plasmids, vectors derived from yeast chromosomes, or from viruses such as baculovirus, papovirus or SV40, retrovirus, adenovirus, adeno-associated virus, or from combinations thereof, in particular from phagemids and cosmids.In a particular embodiment, the expression vector is a vector that allows expression of baculovirus that can infect insect cells.

[0133] If necessary, the sequence encoding the fusion polypeptide also contains, preferably in its 5' part, a sequence encoding a signal peptide for secretion of the fusion polypeptide. Conventionally, the sequence of the signal peptide is a sequence of 15 to 20 amino acids rich in hydrophobic amino acids (Phe, Leu, Ile, Met and Val).

[0134] The vector contains all the sequences necessary for the expression of the sequence encoding the fusion polypeptide. In particular, the vector contains an appropriate promoter selected depending on the host cell into which the construct will be introduced.

[0135] In the context of the present invention, the term "host cell" means a cell capable of expressing a gene carried by a nucleic acid that is heterologous to the cell and that has been introduced into the genome of said cell by transfection techniques.

[0136] Preferably, the host cell is a eukaryotic cell. The eukaryotic host cell is in particular selected from yeast cells such as S. cerevisiae, filamentous fungal cells such as Aspergillus sp, insect cells such as Drosophila S2 cells or Spodoptera sf9 cells, mammalian cells and plant cells. Mammalian cells that may be cited in particular are mammalian cell lines such as CHO, COS, HeLa, C127, 3T3, HepG2 or L(TK-) cells. In a preferred implementation, said host cell is selected from eukaryotic cell lines, preferably Sf9 insect cells. A method for preparing recombinant dimeric proteins in sf9 insect cells is described in US Pat. No. 7,884,190. Any transfection method known to the skilled artisan for the generation of cells expressing heterologous nucleic acids may be used to carry out step a) of the method of the invention. Transfection methods are described, for example, in Sambrook et al., 2001, "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0137] Alternatively, chimeric constructs can be produced by chemical peptide synthesis. For example, the protein of the present invention can be produced by parallel synthesis of shorter peptides, which are then assembled to obtain the complete sequence of the protein with correct disulfide bridges. For example, the synthesis of IL-2 is illustrated in Asahina et al., Angewandte Chemie International Edition, 2015, Vol.54, Issue 28, 8226-8230, the disclosure of which is incorporated herein by reference.

[0138] In another embodiment, the chimeric protein can be expressed in vivo after administration of a nucleic acid encoding said chimeric protein to a subject. In a preferred embodiment, the nucleic acid is carried by an RNA or viral vector, such as an adenovirus-associated virus (AAV).

[0139] Formulation and Route of Administration Also provided are pharmaceutical compositions comprising a chimeric construct, nucleic acid, vector or protein described herein, preferably in association with a pharma- ceutically acceptable vehicle, carrier or excipient (e.g., in solution, suspension or mixture).

[0140] Suitable excipients include any isotonic solution, saline, buffer solutions, sustained release formulations, etc. Liquid, lyophilized or spray-dried compositions are known in the art and can be prepared as aqueous or non-aqueous solutions or suspensions. Preferably, the pharmaceutical composition includes suitable stabilizers, buffers, bulking agents, or combinations.

[0141] The pharmaceutical composition may further contain other active ingredients or may be administered in combination with other active ingredients.

[0142] The pharmaceutical compositions can be administered using any convenient route, including parenteral, e.g., intradermal, subcutaneous or intranasal, with the subcutaneous route being preferred. Oral, sublingual or buccal administration is also encompassed.

[0143] An example of a formulation suitable for subcutaneous injection is described in International Patent Application WO2017 / 068031.

[0144] Treatment of autoimmune and / or inflammatory disorders The pharmaceutical compositions described herein are useful in methods for treating autoimmune and / or inflammatory disorders, such as: systemic lupus erythematosus, type I diabetes, HCV-associated vasculitis, uveitis, myositis, systemic vasculitis, psoriasis, allergies, asthma, Crohn's disease, multiple sclerosis, rheumatoid arthritis, atherosclerosis, autoimmune thyroid disease, autoinflammatory diseases, neurodegenerative diseases including Alzheimer's disease and amyotrophic lateral sclerosis, acute and chronic graft-versus-host disease, spontaneous abortion and allograft rejection; solid organ transplant rejection, vasculitis, inflammatory bowel disease (IBD), and allergic asthma; spondyloarthritis or ankylosing spondylitis; Sjogren's syndrome, systemic sclerosis, alopecia areata, or ulcerative colitis.

[0145] The pharmaceutical compositions described herein are also suitable in conditions in which activation of Treg tissue regenerative properties is desired, such as muscle disease, neurodegeneration, and infarction of the heart or other tissues.

[0146] In a preferred embodiment, the present invention describes a method for treating autoimmune and / or inflammatory disorders, which comprises administering the composition once or twice a week, or even once or twice a month, preferably by subcutaneous route. In one embodiment, a dosage of less than 30 MIU / day, preferably less than 20 MIU / day, is preferred, advantageously less than 10 MIU / day, or between 1 MIU / day and 8 MIU / day. In another particular embodiment, a dosage between 1 and 5 MIU / day, preferably between 0.1 and 3.5 MIU / day is used.

[0147] Generally speaking, doses that allow a 1.2, 1.5, 2, 3, 4 or 5-fold increase in Treg numbers are preferred. The standard unit of measurement for the amount of IL-2 is the International Unit (IU), which is technically an arbitrary weight, but is an amount that produces a certain biological effect in a particular cell proliferation assay as determined by the World Health Organization (WHO). The reason is that i) the weight varies depending on the exact sequence of the molecule and its glycosylation profile, and ii) it is the activity, not the weight of the molecule, that is important.

[0148] The principle of the International Unit is to precisely provide a standard against which any IL-2 molecule can be compared (regardless of the source of the IL-2 or the sequence of the IL-2, including the wild-type sequence or active mutant sequence).

[0149] In fact, the WHO provides ampoules containing IL-2 molecules, which have been calibrated and serve as a standard for determining the dosage of a given preparation of IL-2 (again, regardless of the source or sequence of said IL-2), defined by its potency. By way of example, to determine the dosage of a given preparation of IL-2, the biological activity of a candidate IL-2 preparation is measured in a standard cell proliferation assay using an IL-2-dependent cell line, such as CTLL-2, and compared with that of the standard. The cells are grown in the presence of different doses of the standard. The dose-response effect of IL-2 has been established, in which the dose of IL-2 is plotted on the X-axis as IU and the measured values ​​of proliferation (pr) are plotted on the Y-axis. If one wishes to determine the activity of any IL-2 product, the product is used to grow IL-2-dependent cells and the proliferation is measured. The pr values ​​are then plotted on the Y-axis and a line is drawn from them parallel to the X-axis. From the intersection of this line with the dose-response line, a line is then drawn parallel to the Y-axis. Its intersection with the X-axis gives the activity of the candidate IL-2 product in IU.

[0150] Any changes to the WHO standard ampoules have no effect on either the International Units of the IL-2 preparation or on the determination of its dosage.

[0151] The first standard (WHO International Standard Code 86 / 504, dated 1987) contained purified glycosylated IL-2 derived from Jurkat cells and was arbitrarily assigned a potency of 100 IU / ampule. As stocks of the first International Standard (IS) were running low, WHO needed to replenish it. WHO provided another calibrated IL-2 ampoule, this time made with E. coli. The second standard ampoule contained 210 IU of biological activity per ampoule. A change in standard ampoule does not mean that the IUs change. Thus, determining the dosage of the test IL-2 preparation will not change whether one uses the first standard ampoule, the second standard ampoule, or subsequent standard ampoules as a reference.

[0152] In one embodiment, chronic administration is implemented, including, for example, administration from once every 3 days to once every 3 months. Such administration series can be repeated as necessary.

[0153] In another embodiment, IL-2 is given every other day for 1-2 weeks in a cycle that can be repeated after a break in administration that may last from 3 days to 3 months, preferably 1 to 4 weeks.

[0154] In another embodiment, the treatment can include a first course, also called an induction course, and a second course, which is a maintenance course.

[0155] In certain embodiments, the treatment comprises at least a first course in which the pharmaceutical composition is administered once daily for at least about 2 or 3 consecutive days, preferably 3 to 7 days, and more preferably 4 to 5 consecutive days, preferably followed by a maintenance dose, for example about 6 days later, or about 1 to about 4 weeks later.

[0156] The maintenance dose is typically administered for at least 1 month, preferably at least about 3 months, and more preferably at least about 6 months. In a preferred embodiment, the maintenance dose is administered for about 3 months to about 12 months, preferably about 6 months to about 12 months.

[0157] In a preferred embodiment, the maintenance treatment consists of administration of the pharmaceutical composition weekly, or once or twice every two weeks, or once a month.

[0158] In a preferred embodiment, maintenance treatment consists of administration of interleukin-2 weekly, once or twice every week or every two weeks, or once a month, for a period of at least one month, preferably from about three months to about twelve months.

[0159] Preferably, the maintenance dosage is substantially the same as the first course dosage, or it may be a lower or higher dose.

[0160] Cancer Treatment The pharmaceutical compositions described herein are useful in methods of treating cancer. In some embodiments, the subject is afflicted with locally advanced or metastatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon, lung, ovarian, gastric, bladder, pancreatic, endometrial, breast, renal, esophageal, or prostate cancer. In one embodiment, dosages of less than 30 MIU / day, preferably less than 20 MIU / day, are preferred, advantageously less than 10 MIU / day, or between 3 MIU / day and 5 MIU / day. In other embodiments, 400,000-750,000 IU / kg or 550,000-750,000 IU / kg, preferably 600,000-700,000 IU / kg of IL2 are administered. Dosages may be similar to those prescribed for PROLEUKIN®, but are expected to be less than that.

[0161] The composition may be administered once, from once or more per day to once or more per week; including once every other day.Those skilled in the art will understand that certain factors, including but not limited to, the severity of disease, previous treatment, the overall health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat the subject.Furthermore, the treatment of the subject may include a single treatment or a series of treatments.

[0162] The above protocol examples related to autoimmune and / or inflammatory disorders can be applied in the same or similar manner for use in treating cancer.Alternatively, in another example, the composition can be administered every 8 hours for 5 days, followed by a rest period of 2-14 days, for example 9 days, followed by an administration every 8 hours for another 5 days.In some embodiments, administration is performed in 3 doses every 4 days. EXAMPLES

[0163] Example 1: In vitro and in vivo targeting material and method Fusion proteins Lentiviral vectors were used to generate IL-2 fusion proteins and target-directed proteins. Briefly, human IL-2-C4bpβ fused to scFvE06, which can bind to phosphocholine oxidized lipids (IL-2-C4bpβ-E06 or IL-2N88R-C4bpβ-E06), was integrated into a lentiviral plasmid under the spleen-limited focus-forming virus (SFFV) promoter. "IL-2-C4bpβ-E06" (SEQ ID NO: 19) is human IL-2 fused to the N-terminus of C4BPβ, which is fused to the N-terminus of scFVE06. "IL-2N88R-C4bpβ-E06" (SEQ ID NO: 20) is a mutant IL-2 fused to the N-terminus of C4BPβ, which is fused to the N-terminal region of scFVE06.

[0164] HEK 293T cells were transfected with lentiviral plasmids at 70% confluence using polyethylenimine (PEI) and cultured in serum-free medium for 24–30 h. The supernatants were then filtered, concentrated by ultracentrifugation, resuspended in the appropriate buffer, and subsequently stored at -80 °C. To obtain stable transfected cells, HEK 293T cells were infected with lentiviruses at various multiplicities of infection (MOI) and subsequently sorted for GFP+ cells to ensure that 100% of cells produced IL-2 fusion protein. Stable cell lines were cultured in serum-free medium for 48 h, and the supernatants were collected and purified using HisPur Ni-NTA Resin (Thermofisher), which allows for effective immobilized metal affinity chromatography (IMAC) purification, followed by concentration, buffer exchange, and validation by SDS-PAGE.

[0165] Human pSTAT5 analysis Human blood samples from healthy volunteers were obtained from the Etablissement Francais du Sang (EFS), Saint Antoine Hospital, Paris, France. Informed consent was obtained from each volunteer. The effect of human IL-2, fusion proteins and targeted proteins on the induction of STAT5 phosphorylation (pSTAT5) was evaluated in human CD4+ regulatory T cells (Treg; CD4+Foxp3+CD127lo / -), CD4+ conventional T cells (Tconv; CD4+Foxp3-), CD8+ T cells and natural killer cells (CD3-CD56+) using flow cytometry. Ten-fold dilutions of human IL-2, fusion proteins and targeted proteins mixed with 100 μl of whole blood for 15 min at 37°C were performed before pSTAT5 staining using a Phospho-Epitopes exposure kit (PERFIX EXPOSE kit, Beckman Coulter). In combination with intracellular pSTAT5 signaling, surface markers allow for comparison of activity and median effective concentrations (EC 50 ) values ​​can now be calculated.

[0166] ELISA For detection of targeted proteins, plasma and protein levels were measured following the same protocol as described herein above, except that capture anti-IL-2 monoclonal antibody (MQ1-17h12) or phosphocholine-BSA (LGC Biosearch technology) was coated at 1 μg / mL and detection was performed using anti-His antibody (1:1000; Thermofisher) at 1 μg / mL, followed by development with ultrasensitive streptavidin-HRP (1:2000; Sigma Aldrich).

[0167] Flow cytometry and antibodies Whole blood: After mouse blood lysis, isolated immune cells were stained for 20 min at 4°C with the following antibodies at the optimal dilutions: CD3-PEfluor610 (Invitrogen), CD4V500 (BD Bioscience), CD8AF700 (BD Pharmingen), CD25PeCy7 (Invitrogen), NKp46eF660 (eBioscience), and CD19eF780 (eBioscience). Intracellular detection of Foxp3-FITC (eBioscience) was performed on fixed and permeabilized cells using the FoxP3 staining buffer kit (eBioscience FoxP3 / Transcription). Cells were acquired with a cytoflex S (Beckman Coulter) and analyzed with FlowJo software. Dead cells were excluded by forward / side scatter gating. CD4+ Tregs were defined as CD4+CD25+Foxp3+ cells, CD4+ Teffs as CD4+CD25+Foxp3- cells also called Tconv CD4+CD25+, CD8+ Tregs as CD8+CD25+Foxp3+ cells, CD8+ Teffs as CD8+CD25+Foxp3+ cells also called Tconv CD8+CD25+, natural killer cells as Nkp46+ cells and B cells as CD19+ cells.

[0168] For STAT5 assays, human whole blood was incubated for 15 min with the following antibodies against membrane proteins: CD3-FITC, CD127-PC7, CD4-PB and CD8-KO, CD45RA-AF700 and CD56-APC-eFluor780. After fixation and permeabilization, cells were stained with intracellular p-STAT5-PE and Foxp3-APC antibodies.

[0169] Experimental model of colitis inflammation C57BL / 6 mice were immunized by oral administration of 2.5% dextran sulfate sodium (DSS; Sigma) in drinking water for 6 days. Mice were monitored daily for 10 days for weight, consistency, and the presence of blood in their stool. The following scoring system has been used to assess severity: - Weight loss between 0-5%: 1 / between 5-10%: 2 / between 10-15%: 3 / and over 15%: 4. - Stool form: normal: 0 / formed but soft: 1 / loose: 2 / mild diarrhea: 3 / watery diarrhea: 4. - Gross bleeding: Absent: 0 / Present: 2 / Gross bleeding: 4.

[0170] result The ability of a target-directed fusion protein to recognize its target, dimerize, and maintain its biological activity in vitro The IL-2 construct was conjugated with a tissue-selective moiety to target inflamed tissues without altering its function. N88R -C4bpβ was fused at the C-terminal portion of C4bpβ to scFvE06, which is known to be specific for oxidized phospholipids such as phosphocholine (PC) (Figure 1A).

[0171] To determine the in vitro binding of the targeted proteins, PC-BSA, anti-hIL-2 antibody or BSA were coated, followed by addition of either the targeted proteins or scFvE06 supernatant and development using anti-His-tag antibody (Figure 1B). After PC-BSA coating, the three constructs were detected, meaning that the proteins could bind to PC-BSA. In addition, after hIL-2 coating, only the targeted fusion proteins were detected, highlighting the good conformation of the targeted proteins recognized by anti-IL-2 and anti-His antibodies. Finally, no detection of the targeted proteins was observed after BSA coating, confirming the absence of nonspecific binding and that scFvE06 is specific to PC.

[0172] After filtration, purification and concentration, the recombinant target-directed proteins were similarly characterized after SDS-PAGE followed by Coomassie Blue staining and Western blotting (Figure 1C). A unique signal was detected after Coomassie staining for each protein around 98 kDa, which means that only one major protein was present in the sample under non-reducing conditions (Figure 1C). Regardless of whether the recombinant target-directed proteins were revealed by primary anti-human IL-2 or primary anti-histidine antibodies, under heating and reducing conditions, the IL-2-C4bpβ-E06 and IL-2-C4bpβ-E06 proteins were characterized for their monomeric (50 kDa) and dimeric (100 kDa) forms. N88R A band is observed for -C4bpβ-E06. In addition, a single band at 25 kDa is also observed for each protein after primary anti-histidine antibody development characteristic of scFvE06, regardless of condition. By comparison, native human IL-2 is revealed using only the primary anti-human IL-2 antibody, but in this case a band at 17 kDa is observed.

[0173] Again, a human whole blood assay measuring STAT5 phosphorylation was performed to determine the effect of the fusion proteins targeted to various cell types expressing the IL-2 receptor. 50 The results were approximately 5ng / mL for native hIL-2, 13ng / mL for IL-2-C4bpβ-E06, and IL-2 N88R and 650 ng / mL for IL-2-C4bpβ-E06, which is 3-fold higher than IL-2-C4bpβ-E06 and 130-fold higher than IL-2 N88R This means that -C4bpβ-E06 was required to obtain an equivalent pSTAT5 response on Treg cells (Figure 1D). Differences in pSTAT5 profiles were also observed in Tconv, CD8+ and NK cell populations.

[0174] Indeed, a reduction in pSTAT5 responses is observed with fusion proteins directed against targets at doses up to at least 1000 ng / mL, whereas responses are obtained from doses of 100 ng / mL with native hIL-2 on these effector compartments. For example, less than 20% of Tconv cells, CD8+ cells and NK cells respond to IL-2. N88R -C4bpβ-E06 were activated in response to approximately 10,000 ng / mL. These results highlight the Treg selectivity of the targeted fusion protein due to the reduced binding affinity to the dimeric receptor.

[0175] Demonstration of efficacy of targeted fusion proteins in experimental models of colitis inflammation The targeted fusion proteins were evaluated for their ability to control disease severity in an experimental model of colitis inflammation (Figure 2.A). To obtain similar proliferation of Tregs, the rAAV dose was increased from 5.10 to 10.0 mg / kg in the IL-2-treated group. 10 From rAAV vg, scFvE06, IL-2-C4bpβ-scFvE06 and IL-2 N88R -5.10 for the C4bpβ-scFvE06 treatment group 11 Up to rAAV vg was also applied.

[0176] Seven days after injection, Treg cells expanded and were activated by 2.5-3 fold in the groups treated with rhIL-2 or the targeted fusion protein, as seen by a 3-4 fold increase in CD25 MFI (Figure 2B). The mutant proteins did not expand the effector population, whereas hIL-2 and IL-2-C4bpβ-scFvE06 treatment led to a 3-fold expansion of the effector population. In addition, NK cells expanded only in mice treated with hIL-2, even though the dose was 10 times lower.

[0177] As expected, 5.10 11 No significant changes were observed for these four parameters after rAAV vg scFvE06 injection.

[0178] Six days after DSS administration, untreated mice developed significant clinical symptoms, in this case a significant loss of approximately 10% of their initial body weight associated with mild diarrhea (stool score 3.5) and the presence of blood in the stool explained by severe colonic inflammation (Figure 2C, D, E).

[0179] The Disease Activity Index (DAI), corresponding to the addition of these symptoms, increased from day 3 to day 10, reaching a score of 8 out of 12, and then decreased until day 14 (Figure 2.F). In comparison, mice treated with scFvE06 had similar weight loss and stool form, but little bleeding, explaining the slight decrease in DAI over time compared to untreated mice. Treatment with hIL-2 partially controlled the clinical symptoms, with weight loss, very soft but not watery stool, and little bleeding, explaining the maximum DAI score of 5 10 days after the first DSS administration. Interestingly, mice treated with the targeted fusion protein had a significant decrease in intestinal clinical symptoms, with formed and partially soft stool (maximum score 2) and little bleeding. Finally, the DAI never exceeded a score of 4 in each of the targeted fusion groups, confirming the therapeutic efficacy of these two proteins in treating colitis.

[0180] Interestingly, in the brachial and para-aortic lymph nodes, Tregs expressing integrin α4β7, which is required for crossing the intestinal barrier, were present in a better proportion after treatment with the targeted fusion proteins than with native IL-2. These observations suggest that the number of Tregs recruited to the site of inflammation is improved in the targeted protein group and, indirectly, that the amount of IL-2 in the intestine of mice treated with these targeted proteins is higher, thus suggesting that the combination of IL-2-C4bpβ-scFvE06 and IL-2 N88R The targeting ability of -C4bpβ-scFvE06 is highlighted. Similarly, more Tregs express Ki67 in these two groups compared to native IL-2, which means that these Tregs proliferate more in these two groups.

[0181] [Example 2] In vivo pharmacokinetics method PK analysis Seven-week-old female C57B1 / 6 mice were subcutaneously injected with 100 μl of human IL-2 or IL-2-C4bpβ-scFvE06 50KIU. 100 μl of blood was collected at various time points: baseline, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h and 48 h for hIL-2 dosing by ELISA. Plasma samples were serially diluted 10-fold to 1 / 1000 in the appropriate buffer. Elisa was performed using the supplier's recommendations and plates were read at 450 nm. hIL-2 concentrations in both groups were calculated using the appropriate standard curve.

[0182] In vivo binding of IL-2-C4bpβ-scFvE06 Psoriasis Model The mouse psoriasis model was induced by daily application of 5% imiquimod cream to the ears of mice for 6 days. As an internal control, petrolatum was applied to the contralateral ear of each mouse according to the same schedule. Ten days before psoriasis induction, 8-week-old female Balb / c mice were injected with 5.10 mAb encoding IL-2-C4bpβ-scFvE06. 11 vg AAV was injected IP. Psoriasis control mice were not treated. At the end of the experiment, 6 days after the first application, mice were euthanized, ears were removed and immediately frozen in OCT. Ears were then cross-sectioned at 8 μm using a cryostat, followed by fixation, permeabilization and staining with DAPI (nuclear stain) and anti-6X-HisTag-Cy5 for IL-2-C4bpβ-scFvE06 detection. Sections were analyzed using a fluorescent microscope.

[0183] DSS-induced colitis model DSS-induced colitis model in mice was induced by adding 3% dextran sodium sulfate in drinking water for 6 days. Ten days before colitis induction, 8-week-old female C57B1 / 6 mice were IP injected with 5.1011 vg of AAV encoding either IL2 or IL-2-C4bpβ-scFvE06. A group of control mice was injected but not treated. At the end of the experiment, 10 days after induction, at the peak of inflammation, mice were euthanized and colons were removed and immediately frozen in OCT. Ears were then cross-sectioned at 8 μm using a cryostat, followed by fixation, permeabilization and staining with anti-IL2-HRP or anti-6X-HisTag-HRP for IL-2-C4bpβ-scFvE06 detection. Histochemical brown / black staining was obtained after incubation with Metal enhanced DAB substrate.

[0184] result Pharmacokinetics To determine if we have extended the half-life of the molecules, pharmacokinetic experiments were performed in mice after a single injection of IL2 or IL-2-C4bpβ-scFvE06 at 50kIU SC injection (Figure 3). IL2 increased rapidly with Tmax reaching after 1 hour and then dropped very rapidly until 10 hours, when IL2 was barely detectable. IL-2-C4bpβ-scFvE06, on the other hand, increased more slowly, reaching Tmax between 1 and 4 hours after injection. The plasma concentration also dropped more slowly, and the molecule was still detectable at 24 hours. This result demonstrates that IL-2-C4bpβ-scFvE06 has an extended half-life, calculated to be about 3-fold in this setting. This may extend the duration of the effect of IL-2 on Tregs and maintain the increase in Tregs for a longer period.

[0185] Proof of concept for in vivo targeting of IL-2-C4bpβ-scFvE06 We have developed two models of inflammatory diseases, psoriasis and colitis.

[0186] Psoriasis was induced by topical application of 5% imiquimod cream to one ear, while the contralateral ear was used as an internal control for inflammation and was applied with petrolatum. In these conditions, one ear should develop inflammation that is targeted by IL-2-C4bpβ-scFvE06, whereas the other ear should not develop inflammation and should not be specifically targeted. Briefly, 6 days after daily application of imiquimod and petrolatum, mice were euthanized and ears were removed, frozen, cross-sectioned, and stained with DAPI and anti-6X-HisTag conjugated to Cyanine 5 Fluorochrome for fluorescence microscopy. While untreated control mice did not show any 6X-HisTag staining in either the Vaseline or Imiquimod-treated ears, we observed staining of our molecule throughout the inflamed ear in mice injected with AAV encoding IL-2-C4bpβ-scFvE06. As a control in the same mice, IL-2-C4bpβ-scFvE06 was not detected in the Vaseline-treated ear, demonstrating specific binding of our targeted molecule to inflamed tissue in vivo.

[0187] We further developed a DSS-induced colitis model to test our molecules in various conditions (Figure 4). To induce colitis, 3% DSS was added to drinking water for 6 days. On day 10, when inflammation was at its maximum, mice were euthanized and colons were removed, frozen, cross-sectioned, and stained with anti-hIL2 or anti-6X-HisTag, both conjugated to HRP. Immunohistochemical staining was performed using Metal enhance DAB substrate, which produced black or dark brown color where the molecules were found. As a result, neither the untreated control group nor the IL2-treated group demonstrated detection of IL2 or 6X-HisTag in the colon. However, mice treated with IL-2-C4bpβ-scFvE06 showed detection of both IL2 and 6X-HisTag in the colon, again demonstrating specific binding of IL-2-C4bpβ-scFvE06 in vivo under inflammatory conditions.

Claims

1. i) at least one interleukin 2 (IL2) moiety; ii) at least one targeting moiety that binds to an oxidized protein or an oxidized lipid; and A chimeric construct comprising:

2. The chimeric construct of claim 1 , wherein the targeting moiety binds to a pro-inflammatory oxidized protein or oxidized lipid.

3. The chimeric construct of claim 1 , wherein the targeting moiety binds to an oxidation-specific epitope (OSE).

4. 2. The chimeric construct of claim 1, wherein the targeting moiety binds to (i) a malondialdehyde (MDA) epitope, (ii) a 2-(ω-carboxyethyl)pyrrole (CEP) epitope, (iii) a 4-hydroxynonenal (4-HNE) epitope, or (iv) an oxidized phospholipid (OxPL) such as phosphocholine-containing oxidized phospholipid (PC-OxPL), oxidized phosphatidylethanolamine (OxPE), oxidized phosphatidylserine (OxPS) or oxidized cardiolipin (OxCL), preferably to a phosphocholine-containing oxidized phospholipid (PC-OxPL).

5. 2. The chimeric construct of claim 1, wherein the targeting moiety is an antibody or an antibody fragment such as a single chain variable fragment (scFv), preferably selected from the group consisting of an E06 antibody fragment, such as an E06 antibody or an E06 scFv; an LR04 antibody fragment, such as an LR04 antibody or an LR04 scFv; an NA17 antibody fragment, such as an NA17 antibody or an NA17 scFv; an E014 antibody fragment, such as an E014 antibody or an E014 scFv; an MDA2 antibody fragment, such as an MDA2 antibody or an MDA2 scFv; an IK17 antibody fragment, such as an IK17 antibody or an IK17 scFv; an LR01 antibody fragment, such as an LR01 antibody or an LR01 scFv, and functional variants thereof.

6. The targeting part is - a variable heavy (VH) domain comprising the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 12; and a variable light (VL) domain comprising the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to SEQ ID NO: 11; 6. The chimeric construct of claim 5, which is an E06 scFv or a variant thereof, comprising:

7. 2. The chimeric construct of claim 1, wherein the IL-2 moiety is human IL-2 or a homologous variant thereof, the variant having at least 85% amino acid identity with human wild-type IL-2, preferably the variant is an active analogue of human IL-2 having at least 90% amino acid identity with human wild-type IL-2, and the IL-2 moiety is preferably an IL-2 mutein comprising a substitution at position N88 of SEQ ID NO:2, more preferably the substitution N88R or N88D.

8. 2. The chimeric construct of claim 1, wherein the IL2 moiety and the targeting moiety are fused in frame or via an amino acid linker, preferably a poly-G linker.

9. 2. The chimeric construct of claim 1, wherein the chimeric construct further comprises the beta chain of C4b-binding protein (C4BPβ) or at least one fragment or functional variant thereof capable of forming a dimeric protein, preferably wherein the fragment of C4BPβ comprises or consists of amino acid residues 194 to 252 of C4BPβ, or a longer fragment of C4BPβ extending up to amino acid 135 at the N-terminus.

10. The functional variant of C4BPβ includes: a) a modified sequence of a fragment of C4BPβ in which less than 25 percent, preferably less than 10 percent, of the amino acids of the fragment have been truncated or replaced, and in which the cysteines at positions 202 and 216 and at least three amino acids upstream and downstream of each cysteine ​​are conserved; or b) a modified sequence of a fragment of C4BPβ in which the cysteine ​​responsible for dimerization is replaced by an amino acid preferably selected from alanine, valine, phenylalanine, proline, methionine, isoleucine, leucine and tryptophan, and another amino acid of the fragment of C4BPβ is replaced by a cysteine; or c) the sequence of a fragment of C4BPβ modified by the insertion of a sequence heterologous to the beta chain between the cysteines responsible for dimerization; or d) The sequence of a fragment of C4BPβ modified by excising the amino acids between the cysteines responsible for dimerization.

10. The chimeric construct of claim 9, comprising:

11. The chimeric construct of claim 9, wherein the IL-2 moiety is fused at the N-terminus of C4BPβ or said fragment thereof, and the C-terminus of C4BPβ or said fragment thereof is preferably linked to a targeting moiety.

12. A nucleic acid encoding the chimeric construct of any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. 13. A host cell comprising a nucleic acid according to claim 12 or a vector comprising a nucleic acid according to claim 12.

15. A pharmaceutical composition comprising a chimeric construct according to any one of claims 1 to 11 for use in treating an autoimmune disease and / or an inflammatory disease.

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