Interleukin-2 for use in the treatment of autism spectrum disorder

Abstract

The present invention relates to the use of IL-2 in the treatment of autism spectrum disorder (ASD) in a subject, and in the prevention of ASD in children by administering IL-2 to a child at risk of ASD or to a pregnant mother at risk of having a child with ASD.

Description

[Technical Field]

[0001] The present invention relates to the prevention and treatment of autism spectrum disorders. [Background technology]

[0002] Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by restricted and repetitive behaviors as well as deficits in social communication and interaction. In addition to genetic risk factors, the immune system is also an important factor in the development of ASD. Epidemiological studies have highlighted an increased risk of ASD in children whose mothers suffered from infectious or autoimmune disorders during pregnancy. Mechanistically, animal studies have shown that the association between maternal immune activation (MIA) and ASD is supported by the secretion of IL-17α by maternal Th17 lymphocytes, which crosses the placental barrier and binds to IL-17α receptors on fetal neurons, disrupting neurodevelopment.

[0003] Although psychopharmacological options exist for treating non-specific symptoms associated with ASD, such as irritability and hyperactivity, no medications have been consistently shown to reliably improve core symptoms of ASD.

[0004] Furthermore, there are no effective options to prevent the onset or progression of ASD. Summary of the Invention

[0005] The present inventors now propose the use of interleukin-2 (IL-2) for the prevention and treatment of ASD.

[0006] First, we demonstrated the benefits of IL-2 in a preventive setting. In a mouse model of MIA-induced autism, we stimulated maternal Tregs with low doses of IL-2 and evaluated the effects of this preventive treatment on offspring behavior. We showed that maternal IL-2 treatment during pregnancy prevented abnormalities in early communication, repetitive behaviors, and social deficits, reduced isolating behaviors, and increased all types of social approach and contact.

[0007] Second, we showed that administering low doses of IL-2 to the offspring of MIA-induced autism model mice similarly reduced early symptoms of autistic behavior.

[0008] In view of the above, the present invention provides IL-2 for use in treating autism spectrum disorder (ASD) in a subject. In a preferred embodiment, the subject is a child diagnosed with ASD, preferably the child is 1 to 8 years old.

[0009] In another aspect, the present invention provides IL-2 for use in the prevention of autism spectrum disorder (ASD) in a subject, wherein the IL-2 is administered to a subject that is an infant or child, preferably an infant or child at risk of ASD.

[0010] In yet another aspect, the present invention provides IL-2 for use in preventing autism spectrum disorder (ASD) in an infant or child, wherein the IL-2 is administered to a pregnant mother. In a preferred embodiment, the pregnant mother is at risk of having a child with ASD, and optionally, the pregnant mother has an infectious or autoimmune disease or disorder, and / or, optionally, the pregnant mother has already had a child with ASD or has detectable maternal anti-fetal brain antibodies.

[0011] In certain embodiments, IL-2 is conjugated to a PEG moiety, an Fc fragment, or any other moiety that improves the half-life of IL-2 or its diffusion in the central nervous system. [Brief explanation of the drawings]

[0012] [Figure 1]Figures 1A, 1B, and 1C show the effect of low-dose IL-2 administered to pregnant MIA mouse mothers on preventing the disruption of early social communication skills in their offspring. Figure 1A shows the number of vocalizations per 5 minutes in offspring from control mothers ("PBS," n = 32), MIA ASD model offspring ("polyIC," n = 50), offspring from normal mothers receiving IL2 alone ("IL2," n = 17), and MIA ASD model offspring receiving IL2 ("IL2+PolyIC," n = 26). Figure 1B shows vocalization duration in the offspring of the same mice. Figure 1C shows vocalization modulation in the offspring of the same mice. IL2 was administered as aldesleukin at 50,000 U / day between E7.5 and E11.5. ANOVA *<0.05, **<0.01. Data are reported as mean + / - SEM.

[0013] [Figure 2] Figure 2 shows the effect of low-dose IL-2 administered to pregnant MIA mouse mothers on preventing repetitive behavior in offspring during the marble burying test at week 6 (W6). In the marble burying test, 20 marbles are arranged on a clean bedding surface. The number of marbles buried during a 20-min session is scored for offspring from control mothers ("PBS," n = 25), MIA ASD model offspring ("polyIC," n = 17), normal mothers receiving IL2 alone ("IL2," n = 19), and MIA ASD model offspring receiving IL2 ("IL2+PolyIC," n = 19) (see Malkova et al., 2012). IL2 was administered as aldesleukin at 50,000 U / day between E7.5 and E11.5. ANOVA *<0.05, **<0.01. Data are reported as mean + / - SEM.

[0014] [Figure 3]Figure 3 shows the effect of low-dose IL-2 administered to pregnant MIA mouse mothers on the social interactions of offspring in the three-chamber test at week 7 (W7). See Nadler et al., 2004. Indices of social approach preference were determined in offspring from control mothers ("PBS," n = 16), offspring from the MIA ASD model ("polyIC," n = 21), offspring from normal mothers receiving IL2 alone ("IL2," n = 9), and offspring from the MIA ASD model receiving IL2 ("IL2+PolyIC," n = 14). IL2 was administered as aldesleukin at 50,000 U / day between E7.5 and E11.5. ANOVA *<0.05, **<0.01. Data are reported as mean ± SEM.

[0015] [Figure 4] Figures 4A–4F show the effects of low-dose IL-2 administered to pregnant MIA mouse mothers on solitary behavior and various types of social approach and contact at week 8 (W8), as measured using the Live Mouse Tracker developed by de Chaumont et al. (2019). Data are reported normalized to the offspring of control mothers receiving PBS alone, the offspring of the MIA ASD model ("polyIC", n = 12), the offspring of normal mothers receiving IL2 alone ("IL2", n = 10), and the offspring of the MIA ASD model receiving IL2 ("IL2+PolyIC", n = 12). IL2 was administered as aldesleukin at 50,000 U / day between E7.5 and E11.5. ANOVA *<0.05, **<0.01. Data are reported as mean ± SEM.

[0016] [Figure 5]Figure 5 shows the preventive effect of AAV-IL-2 administered to offspring of MIA model mice at week 3 (W3) on repeated behavior in the marble-burying test at week 6 (W6). In the marble-burying test, 20 marbles are arranged on a clean bedding surface. The number of marbles buried during a 20-min session was scored in offspring of control mothers ("PBS-AAV(e)", n = 8), offspring of MIA ASD model mice ("polyIC-AAV(e)", n = 8), offspring of normal mice administered PBS-AAV(IL2) ("IL2", n = 19), and offspring of MIA ASD model mice administered AAV(IL2) ("PolyIC-AAV(IL2)", n = 15) (see Malkova et al., 2012). PolyIC or PBS was administered at E12.5. AAV-IL2 or AAV-(e) was administered at W3. ANOVA *<0.05, **<0.01. Data are reported as mean + / - SEM.

[0017] Detailed description of the invention: Autism spectrum disorder As used herein, the term "autism spectrum disorder" (ASD) is understood to encompass a family of neurodevelopmental disorders characterized by deficits in social communication and interaction and restricted, repetitive patterns of behavior, interests, or activities, as described, for example, in the "American Psychiatric Association; Diagnostic and Statistical Manual of Mental Disorders (DSM-5) Fifth edition" (DSM-5).

[0018] The term "ASD patient" or "ASD subject" refers to a patient or subject who has received a formal diagnosis of ASD or who is suspected of having ASD, i.e., a subject who exhibits behavioral characteristics and clinical signs of ASD but has not yet received formal verification of the diagnosis. Those skilled in the art are familiar with how patients are diagnosed with ASD, particularly idiopathic ASD. For example, those skilled in the art may diagnose a subject with ASD according to the criteria set forth in the "American Psychiatric Association; Diagnostic and Statistical Manual of Mental Disorders (DSM-5) Fifth Edition." Similarly, ASD patients may be diagnosed according to standardized assessment tools, including, but not limited to, DSM IV, ICD-9, ICD-10, DISCO, ADI-R, ADOS, and m-CHAT. In other cases, patients may have an established DSM-IV diagnosis of autistic disorder or pervasive developmental disorder not otherwise specified (PDD-NOS).

[0019] subject As used herein, the term "subject" or "patient" refers to humans, including newborns, infants (typically 2 months to 1 year old), and children (1, 2, 3, 4, 5, 6, 7, 8 to 12, 13, 14, 15, 16, 17, 18, 19 years old, preferably 1 to 10 years old, preferably 1 to 8 years old, and more preferably 1 to 6 years old).

[0020] In a therapeutic setting, the subject is preferably a child diagnosed with ASD, preferably the child is between 1 and 8 years old, more preferably between 2 and 6 years old, or between 1 and 6 years old.

[0021] In a preventative setting, the term "subject" may preferably refer to an infant or child (preferably, the infant or child is at risk for ASD). Preferably, the subject is an infant or child under the age of 4, under the age of 3, or more preferably under the age of 2.

[0022] Infants or children at risk for ASD include those who have a sibling with ASD or who were born prematurely, typically before 37 weeks of gestation, e.g., between 32 and 36 weeks, or between 23 and 32 weeks. Infants or children at risk for ASD also include those with a history of MIA, i.e., infants or children whose mothers suffered from infections during pregnancy or autoimmune or inflammatory diseases, such as those described below. Infants or children at risk for ASD further include those who exhibit genetic mutations or variants associated with an increased risk of ASD, such as mutations in the FMR1 gene.

[0023] In another embodiment, the term " subject " refers to pregnant mothers, particularly pregnant mothers who are at risk of having a child with ASD. Such women who are prone to have a child with ASD include mothers who have ASD themselves, or have a family history of ASD or autoimmune disease, or who have or have had infectious diseases during pregnancy, or who have autoimmune or inflammatory diseases, and / or who have already had a child with ASD, or who have developed maternal anti-fetal brain antibodies.Anti-fetal brain antibodies are also known as "brain-reactive" antibodies, that is, antibodies that recognize antigens in the central nervous system.

[0024] Typical autoimmune or inflammatory disorders include, but are not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriasis, and autoimmune thyroid disease (Chen et al., 2016).

[0025] In certain embodiments, at-risk mothers include pregnant women who experienced a fever episode in the first or second trimester of pregnancy (Antoun et al, 2021), and / or who experienced at least three fever episodes during pregnancy, and / or a fever lasting seven days or more.

[0026] Preventive Settings The term "preventing" or "prevention" as used herein refers to prophylactic treatment by reducing the onset or risk of onset of the disease, particularly in asymptomatic subjects who are "at risk" or have been determined to be susceptible to developing an ASD. The term "preventing" or "prevention" encompasses reducing the onset of at least one symptom of the disease, such as deficits in social communication and interaction, and restricted, repetitive behavioral patterns, interests, or activities.

[0027] Provided herein is a method for preventing autism spectrum disorder (ASD) in a person prone to developing ASD, comprising: i) in a subject who is an infant or a child in need of prophylaxis; and / or ii) to pregnant mothers to prevent ASD in their children; Methods are provided that include administering IL-2. In embodiment (i), IL-2 is advantageously administered to the subject as early as possible, preferably to a newborn, infant or child under the age of 2 years.

[0028] In embodiment (ii), IL-2 is advantageously administered during or beginning the first trimester of pregnancy. A preferred administration course is once-weekly administration for at least one month, preferably at least two months, and more preferably for two to three months. In another preferred embodiment, the prophylactic treatment may comprise a first course, also designated as an induction course, and a second course, which is a maintenance course. In a particular embodiment, the treatment may comprise at least a first course of IL-2 administered once daily for at least about two or three consecutive days, preferably three to seven days, and more preferably four to five consecutive days, preferably followed by a maintenance dose, for example, about six days later or about one to about four weeks later.

[0029] therapeutic setting Provided herein are methods of treating ASD in a subject in need thereof, comprising administering IL2 to the subject, preferably at a low dose as defined below.

[0030] The term "treat" or "treatment" refers to any improvement in a disease, including alleviating at least one symptom or reducing the severity of the disease. Symptoms include lack of social communication and interaction, restricted and repetitive patterns of behavior, interests, or activities.

[0031] In a preferred embodiment, described herein is a method of treating ASD in a subject, comprising administering the composition once or twice a week, or once or twice a month, preferably by subcutaneous route.

[0032] IL-2 As used herein, interleukin-2 (IL-2) includes mammalian wild-type interleukin-2 and its active analogs. Preferably, the IL-2 is human IL-2 or aldesleukin, as defined below.

[0033] Active analogs of IL-2 have been disclosed in the literature. "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. The IL-2 portion of an active variant generally has 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, e.g., mature wild-type human IL-2.

[0034] As used herein, "wild-type IL-2" refers to IL-2, whether natural or recombinant, containing the 133 amino acid sequence typically found in native human IL-2, as 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 without the signal peptide consisting 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.

[0035] As used herein, "IL-2 mutein" refers to a polypeptide in which specific amino acid substitutions have been made in 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 specified.

[0036] In some embodiments, the cysteine ​​at position 125 is substituted with a neutral amino acid, such as serine (C125S), alanine (C125A), threonine (C125T), or valine (C125V).

[0037] For example, removal of O-glycosylation sites results in a more homogeneous product when the active variant is expressed in mammalian cells such as CHO or HEK cells.

[0038] In certain embodiments, the active 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.

[0039] As used herein, active analogs can selectively promote the proliferation, survival, activation, and / or function of T-reg cells. "Regulatory T cells" or "Tregs" are T lymphocytes characterized as CD4+CD25+Foxp3+ cells and have immunosuppressive activity. "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 exert effector functions (such as cytotoxic activity, cytokine secretion, and inhibition of self-recognition).

[0040] By "selectively enhances," we mean that an active analog enhances activity in T-reg cells but has limited or no ability to enhance activity in non-regulatory T cells. Further described herein are assays for screening for active analogs that selectively enhance the proliferation, survival, activation, and / or function of T-reg cells. Methods for determining whether an IL-2 analog is active are available in the art. See, e.g., WO 2016 / 014428. Active analogs are defined as analogs that exhibit the ability to stimulate Tregs, and include analogs that have improved, similar, or reduced ability to stimulate Tregs compared to wild-type IL-2 or aldesleukin (defined below), so long as they do not stimulate Teffs beyond the stimulation of Tregs. Methods for testing whether a candidate molecule stimulates T cells, particularly Tregs, or NK cells, are well known. Analogs can be tested for their ability to stimulate effector T cells (e.g., CD8+ T cells), CD4+ Foxp3+ Tregs, or NK cells. In preferred embodiments, active analogs exhibit reduced ability to stimulate NK cells compared to wild-type IL2 or aldesleukin. Monitoring STAT5 phosphorylation is a simple method to assess the ability of a variant to preferentially stimulate Tregs over Teffs, as described in Yu et al., Diabetes 2015;64:2172-2183. In certain embodiments, analogs are particularly useful when a predetermined level of STAT5 phosphorylation is achieved for Tregs at a dose at least 10-fold lower than for other immune cells, including Teffs.

[0041] The active analog induces signaling events that preferentially induce the survival, proliferation, activation, and / or function of Treg cells. In certain embodiments, the IL-2 analog retains the ability to stimulate the phosphorylation of STAT5 and / or the 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 analog retains the ability to stimulate the transcription or expression of genes or proteins (e.g., FOXP3, Bcl-2, CD25, or IL-10) or proteins that are important for the survival, proliferation, activation, and / or function of Treg cells. In other embodiments, the IL-2 analog exhibits 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 analog exhibits 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 analog retains the ability of wild-type IL-2 to stimulate phosphorylation of STAT5 and / or one or more signaling molecules downstream of IL-2R in Treg cells, yet exhibits inefficient, reduced, or absent phosphorylation of STAT5, AKT, and / or mTOR or other signaling molecules downstream of IL-2R in FOXP3− CD4+ or CD8+ T cells or NK cells. In other embodiments, the IL-2 analog is inefficient or unable to stimulate survival, proliferation, activation, and / or function of FOXP3− CD4+ or CD8+ T cells or NK cells.

[0042] In all cases, these analogs have the ability to stimulate cell lines such as CTLL-2 or HT-2, which can be universally used to determine biological activity. For example, the biological activity of IL-2 was determined using the HT-2 cell line (clone A5E, ATCC® CRL-1841), whose growth is dependent on IL-2. TM) can be measured in a cell-based assay performed at 490 nm. Cell growth in the presence of a range of test interleukin-2 products is compared to proliferation recorded in the IL-2 International Standard (WHO 2nd International Standard for INTERLEUKIN 2 (human, rDNA-derived) NIBSC code: 86 / 500). Cell growth 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 activated, viable cells. Formazan concentration is then measured spectrophotometrically at 490 nm.

[0043] Examples of IL-2 analogs are disclosed, for example, in EP109748, EP136489, US4,752,585; EP200280, EP118617, WO99 / 60128, EP2288372, US9,616,105, US9,580,486, WO2010 / 085495, WO2016 / 164937.

[0044] For example, certain mutations may reduce affinity for the signaling chain of the IL-2 receptor (IL-2Rβ / CD122 and / or IL-2Rγ / CD132) and / or reduce the ability to induce signaling events from one or both subunits of the IL-2 receptor. Other mutations may confer high affinity to CD25 (IL-2Rα). In either case, such mutations define active variants that preferentially induce Treg survival, proliferation, activation, and / or function. This property can be monitored by surface plasmon resonance.

[0045] Specific examples of useful analogs include IL-2 muteins that exhibit at least one amino acid substitution at 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 appears in the mature sequence of wild-type IL-2 of SEQ ID NO:2.

[0046] Preferred IL-2 muteins contain at least one substitution at position D20H, D20I, D20Y, N30S, Y31H, K35R, V69AP, Q74, N88R, N88D, N88G, N88I, V91K, or Q126L.

[0047] 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, or any combination thereof. In some embodiments, these IL-2 mutein molecules also comprise a substitution at position 125 described herein. In some embodiments, the IL-2 mutein molecule is selected from the group consisting of 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, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91 S, I92K, I92R, E95G, and Q126.In some embodiments, the amino acid sequence of the IL-2 mutein molecule comprises the amino acid sequence set forth in the mature IL-2 sequence plus any of the following substitutions: C125A or C125S and 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, R81H ... and differing by one substitution selected from S, 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, V91 S, 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 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.

[0048] These substitutions can 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.

[0049] In some embodiments, the IL-2 mutein contains an N88R or N88D mutation, preferably N88R. In some embodiments, the IL-2 mutein contains a C125A or C125S mutation. These substitutions can be used alone or in combination with each other. In some embodiments, the mutein contains one, two, three, four, five, six, seven, or eight of these mutations. In some embodiments, the mutein contains each of these substitutions.

[0050] In certain embodiments, the IL-2 moiety is aldesleukin. Aldesleukin is the active ingredient in 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): a deletion of the first amino acid (alanine) and a substitution of cysteine ​​at position 125 with serine. Conservative modifications of IL-2 and substitutions at other positions (i.e., those that minimally affect the secondary or tertiary structure of the mutein) are encompassed. Such conservative substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure* 5 (1978) and *EMBO J.* 8:779-785 (1989) by Argos. 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.

[0051] Variants with mutations that disrupt binding to the α subunit of IL-2R are not preferred, as such variants may have a reduced ability to stimulate Tregs.

[0052] In certain embodiments, the properties of IL-2 (stability, specificity, etc.) may be improved through glycosylation, phosphorylation, fusion to another polypeptide or molecule, polymerization, etc., or chemical or enzymatic modification or addition of the IL-2 molecule (including any active analog thereof).

[0053] In certain embodiments, IL-2 is conjugated to a water-soluble polymer such as polyethylene glycol (PEG). Preferred conjugates are described in patent application WO2012 / 065086, in which the conjugate comprises a water-soluble polymer such as PEG covalently attached to an amine group of the IL-2 moiety via a releasable bond.

[0054] In certain embodiments, IL-2 may be mutated at position D109C (the C residue can be attached to a PEG moiety), for example as described in International Patent Application WO2016 / 0025385.

[0055] In another specific embodiment, IL-2 is fused to an immunoglobulin, preferably an IgG, preferably a human IgG, or preferably to the Fc region of an immunoglobulin. Also, specific fusion constructs comprising two IL-2 proteins fused to one immunoglobulin can be used, as disclosed, for example, in WO2014 / 023752 and WO2015 / 118016.

[0056] In another embodiment, IL-2 is fused at the N-terminus of the Fc portion, either directly or preferably via a peptide linker, e.g., a linker of 8 to 12 amino acids, as described, e.g., in International Patent Application WO2016 / 014428.

[0057] In certain embodiments, IL-2 is conjugated to the beta chain of C4b-binding protein (C4BP) or at least one fragment or functional variant thereof capable of forming a dimeric protein as described in International Patent Application WO2021 / 116444.

[0058] In a preferred embodiment, the IL2 moiety is fused to a fragment of the human C4BP β chain comprising or consisting of at least amino acids 194 to 252, or to a longer fragment of C4BP extending at the N-terminus up to amino acid 135. Preferably, the IL2 moiety is fused at the N-terminus of C4BP β or said fragment.

[0059] In a preferred embodiment, the functional variant of C4BP is a) a modified sequence of a fragment of C4BP, in which less than 25%, preferably less than 10%, of the amino acids of the fragment have been removed or substituted, 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 C4BP, 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 is replaced by a cysteine; or c) the sequence of a fragment of C4BP 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 C4BP modified by removal of amino acids between the cysteines responsible for dimerization; may include:

[0060] In a preferred embodiment, a fusion protein is provided in which one IL2 moiety is fused at the N-terminus of C4BPβ or the fragment thereof and another IL2 moiety is fused at the C-terminus of C4BPβ or the fragment thereof. According to such an embodiment, the fusion protein comprises the following sequence from N-terminus to C-terminus: IL2- C4BP IL2. The IL2 moieties and C4BPβ or the fragment thereof can be fused in frame (directly) or via an amino acid linker, preferably a poly-G linker.

[0061] IL-2 may be used alone or in combination with other therapeutically active agents.

[0062] Production method IL2 can generally be produced by recombinant DNA techniques or RNA molecules in suitable expression vectors.

[0063] Expression vector is selected according to the function of the host cell that construct is introduced.Preferably, expression vector is selected from the vector that allows expression in eukaryotic cell, particularly chromosomal vector or episomal vector or virus derivative, particularly from plasmid, yeast chromosome or virus, such as baculovirus, papovirus or SV40, retrovirus, adenovirus, adeno-associated virus or the vector derived from their combination, particularly phagemid and cosmid.In a specific embodiment, the vector that allows expression of baculovirus can infect insect cell.

[0064] If necessary, the sequence encoding the fusion polypeptide preferably also contains, at its 5' portion, a sequence encoding a signal peptide for secretion of the fusion polypeptide. Conventionally, the signal peptide sequence is a sequence of 15 to 20 amino acids, and is rich in hydrophobic amino acids (Phe, Leu, Ile, Met, and Val).

[0065] The vector contains all the sequences necessary for the expression of the fusion polypeptide-encoding sequence, in particular a suitable promoter selected according to the function of the host cell into which the construct will be introduced.

[0066] 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 that cell by transfection techniques.

[0067] Preferably, the host cell is a eukaryotic cell. Eukaryotic host cells are 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 mentioned in particular are mammalian cell lines, such as CHO, COS, HeLa, C127, 3T3, HepG2, or L(TK-) cells. In a preferred embodiment, the host cell is selected from a eukaryotic cell line, preferably Sf9 insect cells. A method for preparing recombinant dimeric proteins in sf9 insect cells is described in U.S. Patent 7,884,190. Any transfection method known to those skilled in the art for producing cells expressing heterologous nucleic acids may be used to carry out step a) of the method. 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.

[0068] Alternatively, IL-2 can be produced by chemical peptide synthesis. For example, the protein can be produced by parallel synthesis of short peptides, which are then combined to produce the complete sequence of the protein with the correct disulfide bridges. Synthesis of IL-2 is described, for example, in Asahina et al., Angewandte Chemie International Edition, 2015, Vol. 54, Issue 28, pp. 8226-8230.

[0069] In vivo expression In another embodiment, IL2 can be expressed in vivo after administering to a subject a nucleic acid encoding the chimeric protein.

[0070] In a preferred embodiment, the nucleic acid is delivered by an RNA or viral vector, such as an adeno-associated virus (AAV), such as AAV8 or AAV9 (see Wang et al., 2019).

[0071] Typically, a recombinant AAV vector comprises an AAV capsid and an expression cassette comprising a promoter and a nucleic acid encoding a desired transgene (herein, IL2).

[0072] Expression can be constitutive or inducible. The promoter can have ubiquitous or, preferably, cell-specific expression, such as the GFAP promoter for expression in brain cells (astrocytes, see Yshii et al., 2022).

[0073] The amount of recombinant vector administered is adjusted by those skilled in the art so that the IL2 transgene is expressed at a blood level that allows for an increase in the Treg / Teff ratio or for stimulation of Treg without substantially stimulating Teff. 10 of viral genome can be administered.

[0074] Dosage and Regimen The dosage is selected to effectively expand and activate Tregs without substantially activating Teffs.

[0075] This is particularly important when the IL2 is wild-type IL2 or aldesleukin, or a mutein that retains some ability to activate Teffs when used at high doses.

[0076] Generally speaking, doses that increase Treg numbers by 1.2-, 1.5-, 2-, 3-, 4-, or 5-fold are preferred. The standard measure of the amount of IL-2 is the International Unit (IU), which, as determined by the World Health Organization (WHO), is not technically a fixed weight but rather an amount that produces a certain biological effect in a particular cell proliferation assay. This is because i) 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. The principle of the International Unit is to provide a rigorous standard against which any IL-2 molecule can be compared (regardless of origin or sequence, including wild-type or active variant sequences).

[0077] In practice, the WHO provides ampoules containing IL-2 molecules that serve as a calibrated control for determining the dosage of a given IL-2 preparation (regardless of the origin or sequence of the IL-2) defined by its potency. For example, to determine the dosage of a given IL-2 preparation, the biological activity of the candidate IL-2 preparation is measured in a standard cell proliferation assay using an IL-2-dependent cell line, such as CTLL-2-2, and compared with the biological activity of the standard. The cells are grown in the presence of various doses of the standard. A dose-response effect of IL-2 is established, with the dose of IL-2 (in IU) plotted on the X-axis and the proliferation index (pr) plotted on the Y-axis. When the activity of an IL-2 product of unknown activity is to be determined, the product is used to proliferate IL-2-dependent cells, and proliferation is measured. The pr value is then plotted on the Y-axis, and a line parallel to the X-axis is drawn from that value. From the point where this line intersects with the dose-response line, a line parallel to the Y-axis is then drawn. Its intersection with the X-axis provides the activity of the candidate IL-2 product in IU.

[0078] Any modification of the WHO standard ampoule does not affect the International Unit or the determination of the dosage of any IL-2 preparation.

[0079] The first reference standard (WHO International Standard Code 86 / 504, dated 1987) contained purified glycosylated IL-2 from Jurkat cells and was arbitrarily assigned a potency of 100 IU / ampule. Because the first international reference standard (IS) stock solution was running low, WHO had to replace it. WHO provided another calibrated IL-2 ampoule, this time produced in Escherichia coli. The second reference standard ampoule contained 210 IU of biological activity per ampoule. A change in reference standard ampoule does not imply a change in IU. Therefore, the determination of the dosage of a test IL-2 preparation remains the same whether the first reference standard ampoule, the second reference standard ampoule, or any subsequent reference standard ampoule is used as the reference.

[0080] In a preferred embodiment, the treatment may include a first course, also referred to as an induction course, and a second course, which is a maintenance course. In certain embodiments, the treatment may include at least a first course of administering the pharmaceutical composition once daily for at least about 2 or 3 consecutive days, preferably 3 to 7 consecutive days, and more preferably 4 to 5 consecutive days, preferably followed by a maintenance dose after, for example, about 6 days or about 1 to about 4 weeks.

[0081] 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 to about 12 months, preferably about 6 to about 12 months.

[0082] In a preferred embodiment, maintenance treatment consists of administering the pharmaceutical composition once or twice a week, or every week or two weeks, or once a month.

[0083] In a preferred embodiment, maintenance treatment consists of administering interleukin-2 once or twice a week, every week or two weeks, or once a month for a period of at least one month, preferably from about 3 months to about 12 months.

[0084] Preferably, the maintenance dose is substantially the same as the dose of the first course, or may be a lower or higher dose.

[0085] In another embodiment, IL-2 is administered every other day for 1 to 2 weeks in a cycle that can last from 3 days to 3 months, preferably 1 to 4 weeks, and can be repeated after discontinuation of administration.

[0086] In certain embodiments, a subject is administered IL-2 as the sole active ingredient effective to treat ASD or a symptom thereof.

[0087] Typically, the dosage according to the present invention is low, for example, less than 3.5 million IU / day, more preferably less than 3.0 million IU / day, even more preferably less than 2.5 million IU / day, and even more preferably less than 2.0 million IU / day. This dosage effectively activates Tregs without substantially activating Teffs. As a result, the Treg / Teff balance in the subject is dramatically increased. At this dosage, IL-2 significantly induces Tregs while substantially avoiding side effects.

[0088] According to the present invention, IL-2 is administered at a dose preferably in the range of about 0.1 to 3 MIU / day, preferably in the range of 0.5 to 2.5 MIU / day, more preferably in the range of 1 MIU / day to about 2 MIU / day. In preferred embodiments, which are particularly advantageous for subcutaneous administration, IL-2 is administered at doses of 1 MIU / day, 1.5 MIU / day or 2 MIU / day.

[0089] Exemplary dosages are 0.1 to 3 MIU, preferably 0.1 to 1.5 MIU, and more preferably 0.25 to 1 MUI. Preferred dosages are as follows: 3.5 MIU / day, 3.0 MIU / day, 2.5 MIU / day, 2.0 MIU / day, 1.5 MIU / day, 1.0 MIU / day, 0.5 MIU / day, ·0.3MIU / day, 0.1 MIU / day, 0.05 MIU / day, 0.02 MIU / day, or 0.01 MIU / day.

[0090] These dosages may be combined depending on the subject and the progression of the disease.

[0091] The effective dosage can be adjusted by the practitioner based on the profile and age of the subject to which IL-2 is administered, particularly if the subject is a newborn or infant. Typically, IL-2 is administered at a dose of about 0.05 MIU / m 2 / day ~ approx. 2MIU / m 2 / day, preferably 0.2 MIU / m 2 / day ~ approx. 1MIU / m 2 It is administered at a dose of / day.

[0092] The amount of IL-2 so administered preferably depends on the body surface area of ​​the subject. Body surface area (BSA) is the measured or calculated surface area of ​​the human body.

[0093] Various calculations have been published to arrive at BSA without direct measurement. Dubois & Dubois formula (1916):

number

[0094] Another commonly used formula is the Mosteller formula (1987), proposed for use by the Pharmacy and Therapeutics Committee of the Cross Cancer Institute, Edmonton, Alberta, Canada:

number

[0095] More particularly, it is used in children.

[0096] The average BSA for adults is generally 1.73m 2 It is considered to be. TIFF2025530309000003.tif48155

[0097] According to the present invention, treatment (prophylactic or curative) typically includes at least a first course in which interleukin-2 is administered once daily for at least about 2 or 3 consecutive days, preferably 3 to 7 consecutive days, more preferably 4 to 5 consecutive days, followed preferably by a maintenance dose after about 5 or 6 days or about 1 to about 4 weeks.

[0098] 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.

[0099] In a preferred embodiment, maintenance treatment consists of administration of interleukin-2 once or twice a week or every one or two weeks.

[0100] In a preferred embodiment, maintenance treatment consists of administering interleukin-2 once or twice a week, weekly or every two weeks for a period of at least one month, preferably from about three to about twelve months. Preferably, the maintenance dose may be substantially the same as or a lower or higher dose than the first course dose.

[0101] In a preferred embodiment, the treatment comprises at least a first course of interleukin-2 administered once daily for 2 days or 3 to 7 days, preferably 5 days, at a dose of up to 3.5 MIU / day, preferably about 1 to about 2 MIU / day, preferably 1 to 1.5 MIU / day, followed 1 to 2 weeks later by a maintenance dose of about 1 to about 2 MIU / day, preferably 1 to 1.5 MIU / day, administered every 2 weeks for at least 3 months, preferably 6 months.

[0102] In another embodiment, the regimen is adjusted to maintain Treg expansion at at least 1.2, 1.5, 1.7, 1.9, or at least 2-fold the baseline Treg level during maintenance, particularly when the IL-2 molecule is a variant with a longer half-life and / or is conjugated to a moiety that improves the half-life of the conjugate. To that end, in one embodiment, the regimen can be defined as a first (induction) course consisting of a dose of up to 3.5 MIU once daily for 1-3 days, followed by a maintenance course 1-4 weeks later. The maintenance course then preferably consists of administration of the maintenance dose of IL-2 once every 2 weeks to once monthly for approximately 1 month, preferably 3 months, and more preferably 6 months or longer.

[0103] This treatment is typically repeated, i.e., the subject is given several doses of the low dose IL-2 described above, progressively achieving the most substantial benefit. The effectiveness of the treatment is monitored by measuring Tregs, and the dose and administration schedule can be adjusted accordingly.

[0104] The maintenance dose can be administered 2 to 8 weeks after the initial cycle is completed. Preferably, the maintenance dose is the same as the initial dose.

[0105] In certain embodiments, particularly when the subject is a neonate, infant, or child, the method provides a dose of 0.2 MUI / m 2The present invention includes administering at least a first course of IL-2 at a dose of 0.2 MUI / m once daily for at least 3 consecutive days, preferably 3 to 7 consecutive days, and more preferably 4 to 5 consecutive days, followed 1 to 3 weeks later by administering 0.2 MUI / m 2 A maintenance dose is administered below this point, and the administration of the maintenance dose may be repeated, for example, every 1 to 3 weeks.

[0106] Dosage forms and routes of administration IL-2 can be administered using any convenient route, including parenteral, e.g., intradermal, subcutaneous, or intranasal, with the subcutaneous route being preferred, as well as oral, sublingual, or buccal administration.

[0107] IL-2 is typically administered in combination with a pharmaceutically acceptable vehicle, carrier, or excipient (e.g., in a solution, suspension, or mixture). Suitable excipients include any isotonic solution, saline solution, buffered solution, sustained-release formulation, and the like. Liquid, lyophilized, or spray-dried compositions containing IL-2 or its analogs are known in the art and can be prepared as aqueous or non-aqueous solutions or suspensions. Preferably, the pharmaceutical composition includes a suitable stabilizer, buffer, bulking agent, or combination.

[0108] An example of an IL-2 formulation suitable for subcutaneous injection is described in International Patent Application WO2017 / 068031.

[0109] The examples illustrate the invention without limiting its scope. [Example]

[0110] Example 1: Preventive Settings Mouse models of ASD In an established rodent model of maternal immune activation (Smith et al., 2007), offspring from pregnant mice injected intraperitoneally with synthetic dsRNA [poly(I:C), also known as PolyIC], a mimic of viral infection, exhibit behavioral symptoms reminiscent of ASD: social deficits, abnormal communication, and repetitive behaviors (Malkova et al., 2012).

[0111] protocol Pregnant female mice were treated with IL-2 (aldesleukin, 50,000 IU / day subcutaneously) or PBS (control) from E7.5 to E11.5, and then injected IP with poly(I:C) at E12.5 (5 mg / kg).

[0112] Next, we assessed the reversibility of autistic symptoms using various techniques: (i) ultrasonic vocalization recording at D7 to measure early social communication abnormalities, (ii) the marble burying test at W6 to measure restricted and stereotyped behaviors, and (iii) the three-chamber test at W7 to measure desire for social interaction.

[0113] Finally, in W8, we used the Live Mouse Tracker (LMT) to monitor various parameters of social interactions and assess long-term behavior (>14 h of free interaction) using machine learning algorithms, as described in de Chaumont et al., 2019. This protocol analyzes the behavior of mice housed in groups of up to four in an enriched environment in real time over several days. The method combines computer vision via a depth-sensing infrared camera, machine learning for animal and posture identification, and radio frequency identification to monitor the quality of mouse tracking. The system detected up to four animals (two control and two experimental conditions) in the same observation area. All raw behavioral data were then analyzed using Python software (lmt-analysis package) to provide a phenotypic profile for each animal.

[0114] result We observed that prophylactic treatment with ld-IL2 during pregnancy increased maternal Tregs and prevented maternal IL-17 secretion associated with poly(I:C) injection (data not shown).

[0115] Next, we observed that prophylactic treatment with ld-IL2 during pregnancy prevented both quantitative and qualitative abnormalities in early communication (see Figures 1A-1C). Maternal Ld-IL2 also prevented repetitive behaviors (Figure 2) and social deficits (Figure 3) in the offspring.

[0116] In the LMT, ld-IL2 reduced solitary behavior and increased all types of social approach and contact (see Figures 4A-4F).

[0117] We further observed that prophylactic treatment with ld-IL2 during pregnancy reversed the decline in Tregs in the offspring (data not shown).

[0118] These results demonstrate that maternal Treg stimulation with low-dose IL-2 prevents the development of ASD in offspring in a mouse model of MIA-induced autism.

[0119] Example 2: Therapeutic Setting Recombinant adeno-associated virus production and in vivo administration Recombinant rAAV8 vectors were generated by triple transfection of human embryonic kidney 293 T cells as previously described (Churlaud et al., 2014). The transgenes used were luciferase (LUC) and mouse interleukin-2 (IL-2) driven by a hybrid cytomegalovirus enhancer / chicken beta-actin constitutive promoter (CAG). Mice were injected with 1 x 10 rAAV (AAV8-CAG-IL2, herein AAV(IL2) or AAV8-CAG-LUC, herein AAV(e)) diluted in 100 μl of 0.1 M phosphate-buffered saline (PBS). 10 The viral genome (vg) was injected intraperitoneally.

[0120] protocol Pregnant female mice were injected with synthetic dsRNA [poly(I:C) (also known as PolyIC) 5 mg / kg] or PBS (control) at E12.5.

[0121] We used ultrasound vocalization recordings to measure early social communication abnormalities at D7 and subsequently confirmed that the offspring of PolyIC mice exhibit autistic symptoms.

[0122] Next, the offspring were administered AAV-IL2 or AAV-(e) at W3, and then subjected to the marble burying test at W6 to measure restricted and stereotyped behaviors.

[0123] At W5, we further observed an increase in Tregs in AAV-IL2-treated offspring (data not shown).

[0124] result We observed that treatment with AAV(IL2) prevented repetitive behavior in ASD offspring (Figure 5).

[0125] References -Antoun et al. Fever during pregnancy as a risk factor for neurodevelopmental disorders: results from a systematic review and meta-analysis Mol Autism. 2021, 12(1):60. doi: 10.1186 / s13229-021-00464-4. -Chen et al, Maternal autoimmune diseases and the risk of autism spectrum disorders in offspring: A systematic review and meta-analysis, Behavioral Brain Research, 2016, 296:61-69. -Churlaud et al. Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer. Clin Immunol 2014; 151:114-26. -De Chaumont et al.. Real-Time Analysis of the Behaviour of Groups of Mice via a Depth-Sensing Camera and Machine Learning. Nature Biomedical Engineering 2019, 3 (11): 930-42. -Dubois & Dubois Arch Intern Med 1916, 17:863 -Estes & McAllister, Immune mediators in the brain and peripheral tissues in autism spectrum disorder, Nat Rev Neurosci, 2015,16(8):469-86. -Estes & McAllister. Maternal immune activation: Implications for neuropsychiatric disorders Science, 2016, 353(6301):772-7. -Malkova et al. Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism. Brain Behav. Immun. 2012, 26:607-616. -Mosteller RD. Simplified calculation of body-surface area. N Engl J Med 1987; 317:1098 -Nadler et al. Automated apparatus for quantitation of social approach behaviors in mice. Genes Brain Behav. 2004;3:303-314. -Smith et al, Maternal immune activation alters fetal brain development through interleukin-6. J Neurosci. 2007, 27:10695-10702. -Wang et al. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 2019, 18: 358-378. -Yshii, et al. Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation. Nat Immunol 2022, 23: 878-891.

Claims

1. A pharmaceutical for treating autism spectrum disorder (ASD) in a subject, comprising interleukin-2 (IL-2).

2. The pharmaceutical composition of claim 1, wherein the subject is a child diagnosed with ASD, and preferably the child is 1 to 8 years old.

3. A pharmaceutical for preventing autism spectrum disorder (ASD) in a subject, comprising interleukin-2 (IL-2), wherein the IL-2 is administered to the subject who is an infant or child, preferably wherein the infant or child is at risk of ASD.

4. A pharmaceutical for preventing autism spectrum disorder (ASD) in an infant or child, comprising interleukin-2 (IL-2), wherein the IL-2 is administered to the mother during pregnancy.

5. 5. The pharmaceutical of claim 4, wherein the pregnant mother is at risk of having a child with ASD, and optionally wherein the pregnant mother herself has ASD or has or has had an infectious disease or an autoimmune or inflammatory disease during pregnancy, and / or optionally the pregnant mother has already had a child with ASD or has anti-fetal brain antibodies.

6. 5. The method of claim 4, wherein IL-2 is administered during the first trimester of pregnancy.

7. 2. The method of claim 1, wherein IL-2 is administered at a maximum dose of 3.5 MIU / day or less, preferably 3 MIU / day or less, more preferably 2 MIU / day or less, and even more preferably 0.1 to 1.5 or 2 MIU / day.

8. 2. The method of claim 1, wherein IL-2 is administered to a child at a dose of 0.1 to 1 MUI / day, or 0.5 to 1 MIU / day.

9. 2. The pharmaceutical composition of claim 1, wherein IL-2 is administered in a first course, preferably consisting of at least one administration daily for 1 to 7 days, followed by a maintenance course at least 5 days later, wherein the maintenance course preferably comprises or consists of at least one administration per week for 1 week to 3 months.

10. 2. The pharmaceutical composition of claim 1, wherein the IL-2 is human IL-2 or aldesleukin or an active analog thereof, wherein the active analog has at least 85% or 90% amino acid identity with human wild-type IL-2 and is capable of activating Tregs, and more preferably, the analog is an IL2 mutein, preferably comprising a substitution at position N88 of SEQ ID NO: 2, more preferably substitution N88R or N88D.

11. 2. The method of claim 1, wherein the IL-2 is conjugated to a PEG moiety, an Fc fragment, or other moiety that improves the half-life of the IL-2 or its diffusion in the central nervous system.

12. 12. The medicament of claim 11, wherein the IL-2 is conjugated to the beta chain of C4b-binding protein (C4BP) or at least one fragment or functional variant thereof capable of forming a dimeric protein, preferably wherein the fragment of C4BP comprises or consists of amino acid residues 194 to 252 of C4BP or a longer fragment of C4BP extending at the N-terminus up to amino acid 135.

13. The pharmaceutical of claim 1, wherein the IL-2 is expressed in vivo after administration of a nucleic acid encoding the IL-2.

14. The method of claim 13, wherein the nucleic acid is delivered by an RNA or viral vector, preferably an adeno-associated virus (AAV).

15. The method of claim 1, wherein the IL-2 is administered by subcutaneous, intramuscular, or intradermal route.

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