Method to treat tauopathies

Abstract

The present invention relates to the treatment of Tauopathies. In this study, the inventors worked on an optimized treatment of Tauopathies, including AD and primary Tauopathies. Previously, the inventors evidenced that Tau pathology is associated with deleterious T-cell-mediated processes that contribute to promote Tau-related detrimental neuroinflammation and cognitive deficits. Considering the unique capacity of immunosuppressive Tregs to inhibit both CD4+ and CD8+ T cell responses, the inventors raise the hypothesis that amplifying Tregs may allow controlling Tau-driven T-cell-mediated detrimental processes in the course of AD and other Tauopathies. They thus evaluated preclinically the impact on disease progression of an optimized IL-2-based Treg-targeting immunomodulatory treatment in the THY-Tau22 mouse model of Tauopathy. They chronically treated THY-Tau22 mice with an optimized IL-2-based treatment, i.e. complexes of IL-2 and anti-IL-2 antibodies (termed herein IL-2C) in order to modulate Tau-associated detrimental T cell responses. Their data supports that this treatment amplifies Tregs more efficiently and selectively than "regular" low dose IL-2 treatment. Furthermore, they hereby showed that IL-2C has a beneficial effect on cognitive deficits since treated THY-Tau22 mice tend to acquire and retain spatial information more potently than untreated littermates. Thus, the invention relates to an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies.

Description

[0001] METHOD TO TREAT TAUOPATHIES

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies.

[0004] BACKGROUND OF THE INVENTION:

[0005] Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the progressive loss of memory and cognitive functions. Its main histopathological hallmarks are the extracellular aggregation of amyloid 0 (A0) peptides in senile plaques, and the aggregation of hyper- and abnormally phosphorylated tau protein into intraneuronal neurofibrillary tangles (NFT). Tau pathology is also involved in other neurodegenerative diseases, namely a major subtype of Frontotemporal Dementias (FTD) associated with Tau aggregation, which are called primary Tauopathies. These pathologies are defined by an atrophy of the frontal and temporal lobes, known as Frontotemporal lobar degeneration (FTLD). FTLD associated with Tau aggregation (FTLD-Tau) represents 50% of all FTLD cases. It encompasses sporadic and inherited primary tauopathies, including progressive supranuclear palsy (PSP), Pick’s disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), FTLD with mutations in the MAPT gene encoding Tau (FTLD-MAPT) and globular glial tauopathy (GGT) (Hartnell IJ et al., Brain 2021). Besides these anatomopathological lesions, AD and primary tauopathies are also associated with a chronic innate neuroinflammation mediated by microglia and astrocytes, which plays an instrumental and complex role in disease pathophysiology, including both beneficial and detrimental effects (Heneka MT et al., Nat Immunol 2015; Leng F et al., Nat Rev Neurol 202; Hartnell IJ et al., Brain 2021). Previous studies from our consortium have shown that neuroinflammation appears early, at a prodromal stage of AD and impacts disease progression. Indeed, slow decliners (patient with a lesser cognitive decline over the study period) tend to have an increased neuroinflammatory response at the beginning, as opposed to fast decliners (Hamelin L. et al., Brain 2016). Further analysis and clustering of AD patients evidenced two distinct dynamic profiles of neuroinflammatory responses, which vary depending on the individual and differentially impact disease progression (Hamelin L. et al., Brain 2018). Besides AD, growing evidence support that complex neuroinflammatory responses are similarly associated with primary Tauopathies and suggest their involvement in disease pathophysiology (Hartnell IJ et al., Brain 2021). However, little is known on the parameters that modulate such neuroinflammatory responses.

[0006] In addition to central innate neuroinflammation, accumulating evidence shows that peripheral adaptive immunity plays a pivotal role in the pathophysiology of AD and other tauopathies. Genome-Wide Association Studies (GWAS) highlight an association between susceptibility to AD and genes involved in T cell responses and antigen presentation (Lambert JC et al., J Alzheimers Dis 2010 ; Lambert JC et al., Nature genetics 2013 ; Gagliano SA et al., Ann Clinic Transl Neurol ; Kunkle BW et al., Nature genetics 2019). This is corroborated by histopathological observations evidencing higher T cell infiltration in the brains of AD patients (Rogers J. et al., Neurobiol Aging 1988 ; Togo T. et al., J Neuroimmunol 2002) and murine models of AD-like pathology (Browne TC et al., J Immunol 2013 ; Stalder AK et al., J Neurosci 2005). The frequency of peripheral AP-specific T cells also increase with age and in AD patients (Monsonego A. et al., J Clin Invest 2003). Furthermore, AD patients display altered intrathecal immunity, including an increased number of activated CD8+ T cells that correlates with clinical and structural markers of AD pathology (Lueg G. et al., Neurobiol Aging 2015). This has been further emphasized by more recent studies, highlighting an increased frequency of clonally expanded CD45RA+ effector-memory CD8+ T cells in the cerebrospinal fluid (CSF) of AD patients (Gate D. et al., Nature 2020). However, the antigen specificity of such T cells remains poorly defined. Despite this growing body of evidence, the true impact of different T cell subsets on AD progression and pathophysiology, including the mechanisms whereby T cell responses vary in relation to pathological deposits, have yet to be further unraveled.

[0007] Most functional studies to date have focused on A0 pathology and highlight both detrimental and beneficial effects of different T cell subsets. While our previous work shows that AP-specific CD8+ T cells do not induce autoimmune neuroinflammation (Rosset MB et al., J Neuroinflammation 2015), other reports suggest that different subtypes of anti-AP CD4+ T cells promote either meningoencephalitis and Ap clearance (Monsonego A. et al., Proceedings of the National Academy of Science 2006), increased Ap deposition and microglial activation with decreased cognition (Cao C. et al., Neurobiol Dis 2009), or reduced synaptic loss and improved cognitive functions (Ethell DW et al., Neurobiol Dis 2006). Of note, we evidenced that regulatory T cells (Tregs), an immunosuppressive T cell subset, critically control the magnitude of anti-Ap CD4+ T cell responses (Toly-Ndour C. et al., J Immunol 2011). Importantly, we further showed that early amplification of Tregs via low dose IL-2 treatment restored cognitive functions in a mouse model of AD-like amyloid pathology, including through the modulation of plaque-associated activated microglia, whereas early depletion of Tregs accelerated the onset of cognitive deficits (Dansokho C. et al., Brain 2016). Our more recent analyses in this model further evidenced that Tregs also contribute to modulate and fine tune the balance of reactive astrocyte subtypes, including proinflammatory C3 -positive astrocytes, which are associated with detrimental neuroinflammatory responses (Stym-Popper et al., J Neuroinflammation 2023). Hence, variations of peripheral Tregs are inversely correlated with disease progression in AD-like amyloid pathology.

[0008] Much less is known regarding the role of T cells in the context of Tau pathology. Similar to AD, GWAS data highlighted an association between T cell responses and FTD pathogenesis, the larger subtype of which are primary tauopathies (Ferrari R et al., Lancet Neurol 2014). Neuropathological studies in AD patients further emphasize a correlation between T cell infiltration and load of pathological Tau but not AB, supporting that T cells infiltration into the brain parenchyma is driven by Tau-related neurodegenerative changes rather than by cerebral amyloidosis (Merlini M. et al., Neurodegener Dis 2018; Zotova E. et al., Brain 2013). This is supported by our preclinical data in a mouse model of Tau pathology (THY-Tau22 mice), which evidenced a correlation between Tau deposition, early innate neuroinflammation and hippocampal CD8+ T cell infiltration. This extends to humans since FTDP-17 patients harboring the same P301L mutation in Tau also display increased parenchymal infiltration of CD8+ T cells compared to age-matched healthy controls. Importantly, total T cell depletion in THY-Tau22 mice abrogated hippocampal CD8+ T cell infiltration, decreased neuroinflammation and prevented spatial memory deficits (Laurent C. et al., Brain 2017). However, no impact on Tau deposition nor phosphorylation was observed (Laurent C. et al., Brain 2017). This suggests that Tau pathology may drive T-cell-mediated deleterious processes, which contribute to promote Tau-related detrimental innate neuroinflammatory responses and cognitive deficits. Using another mouse model, a recent report by the Holtzman lab similarly supported an instrumental role of T-cell infiltration in Tau-related neurodegeneration, further highlighting the clonal expansion of selected T cells, although their antigen specificity remains unknown (Chen X. et al., Nature 2023). Altogether these studies suggest the therapeutic potential of T-cell-targeting immunomodulatory approaches for the treatment of Tauopathies.

[0009] Moreover, some studies support that an immunomodulatory treatment targeting neuroinflammation (for example anti-TREM2 agonistic antibodies) may have opposite effects depending on disease stage and thus in the context of amyloid and / or Tau pathology (see Schlepckow et al, Lancet Neurology 2023 and Carling et al, J Exp Med 2022 - Editorial on Jain et al, J Exp Med 2022). Hence, said studies show that a beneficial immunomodulatory effect in amyloid-related pathophysiology does not allow to anticipate a similar beneficial effect in Tau- related pathophysiology.

[0010] SUMMARY OF THE INVENTION:

[0011] In this study, the inventors worked on an optimized treatment of Tauopathies, including AD and primary Tauopathies. Previously, in a mouse model of AD-like Tauopathy, the inventors evidenced that Tau pathology is associated with deleterious T-cell-mediated processes that contribute to promote Tau-related detrimental neuroinflammation and cognitive deficits. Considering the unique capacity of immunosuppressive Tregs to inhibit both CD4+ and CD8+ T cell responses, the inventors raise the hypothesis that amplifying Tregs may allow controlling Tau-driven T-cell-mediated detrimental processes in the course of AD and other Tauopathies. This hypothesis is contradictory to the teaching of other studies, which suggest that an activating immunomodulatory approach aimed at enhancing peripheral immunity, including effector memory T cells, is beneficial in Tau-related pathophysiology (see Rosenzweig et al, Nature Comm 2019).

[0012] They thus evaluated preclinically the impact on disease progression of an optimized IL- 2-based Treg-targeting immunomodulatory treatment in the THY-Tau22 mouse model of Tauopathy. They chronically treated THY-Tau22 mice with an optimized IL-2 -based treatment, i.e. complexes of IL-2 and anti-IL-2 antibodies (termed herein IL-2C) in order to modulate Tau- associated detrimental T cell responses. Mice were treated from the onset of Tau pathology and neuroinflammation (3 months of age) until behaviour evaluation, around 8.5 to 9.5 months of age. Their data supports that this treatment amplifies Tregs more efficiently and selectively than “regular” low dose IL-2 treatment. Importantly, IL-2C treatment did not significantly impact the frequency of peripheral effector T cells (Teff) nor effector memory T cells (TEM) and resulted in heightened Tregs / Teff as well as Tregs / TEM ratio as compared to “regular” low dose IL-2 treatment. Furthermore, they hereby showed that IL-2C has a beneficial effect on cognitive deficits since treated THY-Tau22 mice tend to acquire and retain spatial information more potently than untreated littermates. Finally, they demonstrated that this effect on spatial memory is associated with the modulation of neuroinflammatory responses.

[0013] The effect of the IL-2C was thus surprising since prior art taught that an activating immunomodulatory approach, which would enhance rather than dampen peripheral immunity, will have beneficial effects in Tau-related pathophysiology (see Rosenzweig et al, Nature Comm 2019). Thus, the invention relates to an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies. Particularly, the invention is defined by its claims.

[0014] DETAILED DESCRIPTION OF THE INVENTION:

[0015] In a first object, the invention relates to an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies.

[0016] According to the invention, the IL-2C acts as a booster of Tregs cells.

[0017] Thus, in a particular embodiment, the invention also relates to a booster of Tregs cells which is an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies.

[0018] In one embodiment of the invention, the complex IL-2C amplifies selectively the pool of Treg cells. In one embodiment of the invention, the complex IL-2C increases the survival of Treg cells.

[0019] In one embodiment of the invention, the IL-2C improves the functionality of Treg cells.

[0020] In another embodiment of the invention, the IL-2C does not amplify effector T cells (Teff).

[0021] In another embodiment of the invention, the IL-2C does not amplify B cells.

[0022] In another embodiment of the invention, the IL-2C increases the ratio Treg / Teff. Within the context of this invention, a boosting of Treg cells refers to any increase in proportion of Treg cells relative to Teffs, in number or in activity as tested by suppressive assays or by expression of molecules that reflect the activity of the Tregs such as CD25, the alpha-chain of the IL-2 receptor, in a subject. The augmentation in proportion is preferably by at least 10%, 20%, 30%, 40%, 50%, 60% as compared to the level prior to treatment. In one embodiment, the boosting designates a shift in the Treg / Teff balance towards Tregs, or an increase in the Treg / Teff ratio.

[0023] The boosting of Tregs can be determined by measuring the number of Tregs (e.g., based on the expression of CD25, Foxp3, CD127) and / or the activity of Tregs in samples from the treated subject. The absence of substantial boosting of Teff can also be determined by measuring the number of Teff and / or the activity of Teff in samples from the treated subject. Preferably, the absence of substantial boosting of Teff indicates the target Teff cell population does not acquire markers of activation such as CD25, CD69 and / or HLA-DR, or as assessed by whole transcriptome analyses. Detailed methods for detecting, measuring and quantifying Treg and Teff cells are well known per se in the art. In one embodiment, the boosting of Treg cells is performed as in Example 3 described here below. In one embodiment, the IL-2C increases circulating CD4+CD25hiCD1271oFoxp3+

[0024] Tregs.

[0025] In one embodiment, the complex IL-2C increases circulating CD8+CD25hiFoxp3+ Tregs.

[0026] As used herein, the term “Tauopathies” has its general meaning in the art and denotes a class of neurodegenerative diseases involving the aggregation of tau protein into neurofibrillary or gliofibrillary tangles in the human brain. Tangles are formed by abnormal and hyperphosphorylation of the microtubule-associated protein known as tau, causing the protein to dissociate from microtubules and form insoluble aggregates. These aggregations are also called paired helical filaments. Tauopathies include but are not limited to primary Tauopathies (FTLD-tau) like progressive supranuclear palsy (PSP), Pick's disease, corticobasal degeneration (CBD), argyrophilic grain disease, globular glial tauopathy, FTLD-MAPT (FTLD with genetic mutations in the gene encoding tau), behavioural variant of Frontotemporal dementia, progressive non-fluent aphasia, semantic dementia, as well as secondary tauopathy like Alzheimer's Disease.

[0027] As used herein, the term “Alzheimer's disease” denotes a progressive cognitive and functional impairments of the brain. Early symptoms can affect the most complex daily living activities. The most noticeable deficit is a cognitive deficit, which shows up as difficulty in remembering recently learned facts and inability to acquire new information. Alzheimer's disease and Alzheimer-related disorders include but are not limited to amyloid-related disorders such as: mild cognitive impairment; prodromal Alzheimer’s disease; early Alzheimer's disease; Alzheimer's disease, including sporadic (non-hereditary) Alzheimer's disease and familial (hereditary) Alzheimer's disease; early onset Alzheimer’s disease (“EOAD”); late onset Alzheimer's disease (“LOAD”); age-related cognitive decline; posterior cortical atrophy (or Benson syndrome); logopenic progressive primary aphasia; cerebral amyloid angiopathy (“CAA”); vascular dementia; hereditary cerebral haemorrhage; Down's syndrome; inclusion body myositis ("LBM"); or age-related macular degeneration (“ARMD”). The symptoms include but are not limited to: memory loss (recent memory, personal history, conversations...), cognitive deficits, behavioural and mood changes, depression, apathy.

[0028] In one embodiment, the Tauopathies are in a chronic phase or in an acute phase.

[0029] In another embodiment, the Tauopathies are at an early stage or at a mild stage or at a moderate stage. In another embodiment, the Tauopathies are at a pre-dementia stage or at a pro-dromal stage or at a stage of mild cognitive impairment.

[0030] In another embodiment, the Tauopathies are at a pre-symptomatic stage.

[0031] Particularly, the invention relates to an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies like Alzheimer's disease and other Tauopathies, by restoring longterm memory and cognitive functions.

[0032] Particularly, the invention relates to an IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies like Alzheimer's disease and other Tauopathies, by decreasing the chronic innate neuroinflammatory response mediated by microglia and astrocytes.

[0033] As use herein the term “Treg cells” refers to a subpopulation of T cells that modulate immune responses, maintain immunological tolerance to self-antigens, suppress excessive immune responses deleterious to the host, and abrogate autoimmune diseases. T regulatory cells are a component of the immune system that suppresses immune responses of other cells. Regulatory T cells come in many forms with the most well -understood being those that express CD4, CD25, and Foxp3.

[0034] As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject according to the invention is a human. Particularly, a subject according to the invention is a human with a Tauopathy.

[0035] As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease, as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse or slowing of disease progression. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness (e.g., the pattern of dosing used during therapy). A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" or “induction phase” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" or “maintenance phase” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g. to keep the subject in remission or in improved conditions for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g. weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, disease worsening, etc.]).

[0036] The invention also relates to a method of treating Tauopathies in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of an IL-2 / anti-IL-2 complex (IL-2C).

[0037] The IL-2C complex

[0038] The IL-2

[0039] As used herein, the term IL-2 refers to the interleukin-2 and denotes a cytokine signalling molecule in the immune system. It is a 15.5-16 kDa protein that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity. IL-2 is part of the body's natural response to microbial infection, and in discriminating between foreign ("non-self ') and "self. IL-2 mediates its effects by binding to IL-2 receptors, which are expressed by lymphocytes. The major sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells. Put shortly the function of IL-2 is to stimulate the growth of helper, cytotoxic and regulatory T cells. A nucleotide and an amino acid sequence of human IL-2 are disclosed, for instance, in Genbank ref 3558 or P60568, respectively.

[0040] In one embodiment and according to the invention, IL-2 of the IL-2C is native or obtained by recombinant or synthetic techniques. IL-2 may be or comprise the native polypeptide sequence or can be an active variant or fragment of the native IL-2 polypeptide. IL-2 is obtained from different sources including mammalian sources such as e.g., human, mouse, rat, primate, and pig.

[0041] In one embodiment of the invention, the IL-2 polypeptide or active variant is derived from a human source, and includes recombinant human IL-2, particularly recombinant human IL-2 produced by microbial hosts. Active variants of IL-2 have been disclosed in the literature. In one embodiment of the invention, IL-2 is a variant of the native IL-2, which can be fragments, analogues, and derivatives thereof. As used herein, the term “fragment” denotes a polypeptide comprising only a part of the intact polypeptide sequence. As used herein, the term an “analogue” designates a polypeptide comprising the native polypeptide sequence with one or more amino acid substitutions, insertions, or deletions. Muteins and pseudopeptides are specific examples of analogues. As used herein, the term “Derivatives” refers to any modified native IL-2 polypeptide or fragment or analogue thereof, 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.). Active variants of a reference IL-2 polypeptide generally have at least 75%, preferably at least 85%, more preferably at least 90% amino acid sequence identity to the amino acid sequence of the reference IL-2 polypeptide. Methods for determining whether a variant IL-2 polypeptide is active are available in the art. An active variant is, most preferably, a variant that boosts Treg cells. Examples of IL-2 variants are disclosed, for instance, in EP109748, EP136489, US4,752,585; EP200280, or EPl 18,617.

[0042] In one embodiment of the invention, IL-2 is a recombinant IL-2, such as for example, an IL-2 that has been prepared by recombinant DNA techniques. The host organism used to express a recombinant DNA encoding IL-2 may be prokaryotic (a bacterium such as E. coli) or eukaryotic (e.g., a yeast, fungus, plant or mammalian cell). Processes for producing IL-2 have been described e.g., in US4,656,132; US4,748,234; US4,530,787; or US4,748,234, incorporated therein by reference. In a preferred embodiment, the invention uses an IL-2 of human origin, or an active variant thereof, more preferably produced recombinantly.

[0043] In one embodiment of the invention, IL2 is a human IL-2. IL-2 for use in the present invention shall be in essentially pure form, e.g., at a purity of 95% or more, further preferably 96, 97, 98 or 99% pure.

[0044] In one embodiment of the invention, the IL-2 is in monomeric or multimeric form.

[0045] IL-2 is commercially available, including for pharmaceutical uses, and it is authorized for use in human patients. Examples of IL-2 include but are not limited to: Proleukin®, Aldesleukin®, Roncoleukin®. Specific examples of IL-2 formulations are described in US4,604,377.

[0046] Where IL-2 is in monomeric form, it is preferred to add to the compositions an amino acid base sufficient to decrease aggregation of IL-2 during storage. The amino acid base can be an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Examples of such amino acids include arginine, lysine, and aspartic acid. In a particular embodiment, the composition comprises a multimeric IL-2, for example lyophilized.

[0047] A specific example of such a composition is Proleukin® IL-2. This lyophilized formulation comprises selectively oxidized, recombinant IL-2 admixed with a water-soluble carrier, such as mannitol, that provides bulk, and SDS to ensure solubility of IL- 2 in water. This composition is suitable for reconstitution in aqueous solutions for parenteral injection.

[0048] The current packaging of IL-2 is vials containing 18 M IU of IL-2. Given the ultra-low dose to be used according to the invention, packaging for dose of 0.01 MIU, 0.02 MIU, 0.5 MIU 0.1 MIU, 0.2 MIU, 0.5 MIU, 1 MIU and 3, or 3.5 MIU are preferably prepared.

[0049] Anti -IL-2

[0050] In one embodiment and according to the invention, the anti-IL-2 of the IL-2C is any antibody that specifically targets defined epitope of IL-2 (notably the human IL-2) to preferentially bind CD25 (i.e. CD25-biased) for selectively stimulating the trimeric IL-2 receptor (consisting of CD25, CD122 and CD132), thus preferentially expanding regulatory T (Treg) cells.

[0051] As used herein the term "antibody" or "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the binding site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L- CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

[0052] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as IL-2, while having relatively little detectable reactivity with non-IL-2 proteins or structures. Specificity can be relatively determined by binding or competitive binding assays, using, e.g. Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g. an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity / avidity in binding to the specific antigen versus non-specific binding to other irrelevant molecules (in this case the specific antigen is IL-2).

[0053] The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

[0054] The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0055] The antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.

[0056] In one embodiment, the monoclonal antibody of the invention is a chimeric antibody, particularly a chimeric mouse / human antibody.

[0057] According to the invention, the term "chimeric antibody" refers to an antibody that comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.

[0058] In some embodiments, the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region that belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).

[0059] In another embodiment, the monoclonal antibody of the invention is a humanized antibody. In particular, in said humanized antibody, the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and nonhuman donor CDRs, such as mouse CDRs.

[0060] In one embodiment, the humanized antibody can be derived from a chimeric antibody.

[0061] In another embodiment, the monoclonal antibody of the invention is a caninized or primatized based on the same methods of humanization.

[0062] According to the invention, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.

[0063] The humanized antibody of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred. Examples of tandem type humanized antibody expression vector include pKANTEX93 (WO 97 / 10354), pEE18 and the like. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0064] In some embodiments the antibody of the invention is a human antibody.

[0065] As used herein the term "human antibody is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0066] Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368-74 (2001) and lonberg, cur. Opin. Immunol. 20; 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, or which are present extra-chromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech. 23;1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application publication No. US 2007 / 0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 13: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86(1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). Fully human antibodies can also be derived from phagedisplay libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581). Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT publication No. WO 99 / 10494. Human antibodies described herein can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

[0067] In one embodiment, the antibody of the invention is an antigen-biding fragment selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VL or VH chains.

[0068] The term “antigen-binding fragment” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen (e.g., IL-2). Antigen-binding functions of an antibody can be conferred by fragments of an intact antibody. Examples of binding-fragments encompassed within the term antigen binding-fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). "dsFv" is a VH:VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. Such single chain antibodies include one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies. A unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies. Antigen-binding fragments can be incorporated into single domain antibodies, SMIP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Antigen-binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

[0069] The Fab of the present invention can be obtained by treating an antibody that specifically reacts with IL-2 with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.

[0070] The F(ab')2 of the present invention can be obtained by treating an antibody that specifically reacts with IL-2 with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.

[0071] The Fab' of the present invention can be obtained by treating F(ab')2 that specifically reacts with IL-2 with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

[0072] The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three-dimensional structure (see, e. g., W098 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0073] Domain Antibodies (dAbs) are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004 / 0110941; EP 1433846, 0368684 and 0616640; WO 2005 / 035572, 2004 / 101790, 2004 / 081026, 2004 / 058821, 2004 / 003019 and 2003 / 002609, each of which is herein incorporated by reference in its entirety.

[0074] UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007 / 059782, which is incorporated by reference in its entirety.

[0075] In one embodiment and according to the invention, the anti-IL-2 of the IL-2C is a single domain antibody.

[0076] As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals, which are naturally devoid of light chains. Such single domain antibody are also called VHH or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484- 490; and WO 06 / 030220, WO 06 / 003388. The nanobody® has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e. camelid nanobodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus, yet another consequence of small size is that a nanobody® can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug rather than that of a classical antibody. The low molecular weight and compact size further result in nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitate drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. The amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs", which are referred to in the art and herein as "Framework region 1" or "FR1 as "Framework region 2" or "FR2"; as "Framework region 3 " or "FR3"; and as "Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http: / / imgt.cines.fr / ).

[0077] Camel Ig can be modified by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "nanobody®" or “VHH”. See U.S. patent number 5,759,808 issued June 2, 1998; see also Stijlemans, B. et al. , 2004 J Biol Chem 279: 1256-1261 ; Dumoulin, M. et al. , 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440- 448; Cortez- Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. In certain embodiments herein, the camelid antibody or nanobody® is naturally produced in the camelid animal, i.e. is produced by the camelid following immunization with IL-2 or a peptide fragment thereof, using techniques described herein for other antibodies. Alternatively, the IL-2-binding camelid nanobody® is engineered, i.e. produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody® proteins using panning procedures with IL-2 as a target.

[0078] In some embodiments, the single domain antibody is a “humanized” single domain antibody.

[0079] As used herein the term “humanized” refers to a single domain antibody of the invention wherein an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional chain antibody from a human being. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be chosen such that the resulting humanized single domain antibodies still retain the favorable properties of single domain antibodies of the invention. The one skilled in the art is able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions.

[0080] Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and / or VL, e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR- mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al. In some embodiments, the glycosylation of an antibody is modified. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0081] In some embodiments, some mutations are made to the amino acids localized in aggregation “hotspots” within and near the first CDR (CDR1) to decrease the antibodies susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647).

[0082] The antibody of the present invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions. IgGl and IgG3 are isotypes that mediate such effectors functions as ADCC (Antibody-dependant cellular cytotoxicity) or CDC (Complement-dependent cytotoxicity), when IgG2 and IgG4 don’t or in a lower manner. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the monoclonal antibodies of the present invention may be changed by isotype switching to, e.g. an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.

[0083] An example of an appropriate anti-IL-2 antibody is disclosed in US20170114130A1.

[0084] In one embodiment and according to the invention, the anti-IL-2 of the IL-2C is a protein or polypeptide that will bind to IL-2.

[0085] Particularly, the anti-IL-2 of the invention recognizes the human IL-2.

[0086] IL-2 / anti-IL-2 complex (IL-2C)

[0087] In one embodiment and according to the invention, the IL-2 / anti-IL-2 complex (IL-2C) comprise the IL-2 and an anti-IL-2 according to the invention and are fused together.

[0088] IL-2 / anti-IL-2 complex is well known in the priori art (see for example Boyman O. et al. Science 2006, Letourneau S. et al. Proc Natl Acad Sci U S A. 2010, VanDyke D. et al. Cell Rep 2022, Spangler J. B. et al. Immunity 2015 or the patent application WO2022038193). Typically, the IL-2 and an anti-IL-2 are fused at their N terminal end or at their C terminal end to provide a fusion protein or fusion complex.

[0089] According to the invention, the fusion protein or fusion complex may be produced by conventional automated synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The complex of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The complex of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides.

[0090] In a particular embodiment, the anti-IL-2 of the IL-2C interacts with a defined epitope of IL-2 amino acid residues (notably the human IL-2) comprising:

[0091] - Hl 6, D20 on the alpha helix;

[0092] - Q57, E60, E61, L63, K64, E67, E68 on the B and B’ helix; and

[0093] - L80, R81, R83, D84, 186, S87, N88, N90, V91, L94, E95, K97, T101, T102, M104 on the C helix and C-D loop.

[0094] Nucleic acid sequence

[0095] Accordingly, a further object of the invention relates to a nucleic acid molecule encoding IL-2 according to the invention, an anti-IL-2 according to the invention and / or an IL- 2 / anti-IL-2 complex according to the invention.

[0096] The nucleic acid of the invention can be DNA or RNA. As used herein, the terms “coding sequence” or “a sequence which encodes a particular protein” or “encoding nucleic acid” or “nucleic acid molecule encoding”, denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0097] Vectors

[0098] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. As used herein, the terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the Introduced sequence. So, a further aspect of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (’’Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, gPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0099] Non-viral vectors

[0100] In a preferred embodiment, the vector use according to the invention is a non-viral vector. Typically, the non-viral vector may be a plasmid encoding a complex according to the invention.

[0101] Particularly, a non-viral vector can be an exosome.

[0102] Non-viral vectors mainly comprise chemical systems that are not of viral origin and generally include chemical methods such as cationic liposomes and polymers. Non-viral vectors useful in the practice of the present invention are very well known in the art. According to the invention, non-viral vectors include but are not limited to liposomes such as cationic liposomes, solid-lipid nanoparticles (SLNs or LNPs) such as [(4- hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2-hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (dLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (dLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM-102-based nanonparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al.Biol Pharm Bull (2018). Non-viral vectors according to the invention include also the non-viral vectors described in patent WO2017049245 and W02018081480.

[0103] In some embodiments, the method according to the invention, wherein the non-viral vector is a cationic polymers-based nanoparticle, and more particularly is a polyethylenimine (PEI)-based nanoparticle.

[0104] In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) encoding a complex according to the invention.

[0105] In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding a complex according to the invention.

[0106] Viral vectors Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.

[0107] The terms “Gene transfer” or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e. g. , episomes), or integration of transferred genetic material into the genomic DNA of host cells.

[0108] Examples of viral vector include adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

[0109] Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95 / 14785, WO96 / 22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056 and WO94 / 19478.

[0110] In a particular embodiment, adeno-associated viral (AAV) vectors are employed.

[0111] In another particular embodiment, the AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO and all variants of AAV9, including AAV PHP.B (see for example the patent application WO2015038958), AAVPHP.eB, AAV-PHP.N", and "AAV-PHP.B- DGT (see the patent application W02017100671 or Chan Y Ken, Nat Neurosci. 2017 Aug;20(8): 1172-1179.), AAV3B, AAV-2i8, Rh74, AAV capBlO, AAVMacPNSl or AAVMacPNS2 or any other serotypes of AAV that can infect human, monkeys or other species.

[0112] By an "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV9 etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g. functional ITRs) of the virus. The ITRs need not be the wildtype nucleotide sequences, and may be altered, e.g. by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (i.e. the CYP46A1 gene) and a transcriptional termination region.

[0113] The control elements are selected to be functional in a mammalian cell. The resulting construct that contains the operatively linked components is bounded (5'and Y) with functional AAV ITR sequences. By "adeno-associated virus inverted terminal repeats " or "AAVITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e. g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds. ) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. Furthermore, 5' and 3'ITRs that flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e. to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. Furthermore, 5' and 3'ITRs that flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

[0114] Particularly preferred are vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian CNS, particularly neurons. A review and comparison of transduction efficiencies of different serotypes is provided in Cearley CN et al., 2009. In one preferred example, AAV-2 -based vectors have been shown to direct long-term expression of transgenes in CNS, preferably transducing neurons. In other nonlimiting examples, preferred vectors include vectors derived from AAVrhlO serotype, which have also been shown to transduce cells of the CNS and particularly deep grey nucleus and diffusely cortex and white matter.

[0115] The selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo. Such control elements can comprise control sequences normally associated with the selected gene.

[0116] Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG, neuronal promoters, promoter of Dopamine- 1 receptor and Dopamine-2 receptor, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e. g. , Stratagene (San Diego, CA). For purposes of the present invention, both heterologous promoters and other control elements, including but not limited to CNS-specific and inducible promoters, enhancers and the like, will be of particular use.

[0117] Examples of heterologous promoters include the CMV promoter. Examples of CNS- specific promoters include those isolated from the genes from myelin basic protein (MBP), glial fibrillary acid protein (GFAP), and neuron specific enolase (NSE).

[0118] Examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia andaufin.

[0119] The AAV expression vector which harbours the DNA molecule of interest bounded by AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e. g. , U. S. Patents Nos. 5,173, 414 and 5,139, 941; International Publications Nos. WO 92 / 01070 (published 23 January 1992) and WO 93 / 03769 (published 4 March 1993); Lebkowski et al., 1988 ; Vincent et al., 1990; Carter, 1992 ; Muzyczka, 1992 ; Kotin, 1994; Shelling and Smith, 1994 ; and Zhou et al., 1994. Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques. AAV vectors which contain ITRs have been described in, e.g. U. S. Patent no. 5,139, 941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC") under Accession Numbers 53222,53223, 53224,53225 and 53226. Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5' and 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian CNS cells (notably neurons or astrocytes) can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e. g., Edge, 1981 ; Nambair et al., 1984 ; Jay et al., 1984. In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e. g. , Graham et al., 1973;, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome mediated gene transfer (Mannino et al., 1988), lipid-mediated transduction (Feigner et al., 1987), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., 1987).

[0120] Delivery of the vectors

[0121] It is herein provided a method for treating Tauopathies in a subject in need thereof, said method comprising:

[0122] (a) providing a vector as defined above, which comprises a complex-encoding nucleic acid; and

[0123] (b) delivering the vector in the periphery and / or in the brain and / or intrathecally.

[0124] According to the invention, the vector can be administrated intravenously, and / or in the brain, and / or intrathecally, simultaneously, separately or sequentially.

[0125] As used herein, the term “simultaneous use” denotes that the intravenous, and / or the intracerebral, and / or the intrathecal administration occur at the same time.

[0126] As used herein, the term “separate use” denotes that the intravenous, and / or the intracerebral, and / or the intrathecal administration does not occur at the same time. As used herein, the term “sequential use” denotes that the intravenous, and / or the intracerebral, and / or the intrathecal administration occur by following an order.

[0127] To deliver the vector specifically to a particular region and to a particular population of cells, the vector may be administered by intracerebral and / or intrathecal injection.

[0128] Additional routes of administration may also comprise local application of the vector under direct visualization, e.g. superficial cortical application, or other non-stereotactic application, including but not limited to either intrathecal injection and / or peripheral administration of blood-brain-barrier-permeant viral vectors.

[0129] Particularly, the method of the invention may comprise intracerebral administration through stereotaxic or neuro-navigated injections. However, other known delivery methods may also be adapted in accordance with the invention. For example, for a more widespread distribution of the vector across the CNS (for an intracerebral injection in the periventricular white matter), it may be injected into the cerebrospinal fluid, e.g. by lumbar puncture. To direct the vector to the peripheral nervous system, it may be injected into the spinal cord or into the peripheral ganglia, or the flesh (subcutaneously or intramuscularly) of the body part of interest. In certain situations, the vector can be administered via an intravascular approach. For example, the vector can be administered intra-arterially (carotid) in situations where the blood-brain barrier is disturbed or not disturbed. Moreover, for more global delivery, the vector can be administered during the "opening" of the blood-brain barrier achieved by infusion of hypertonic solutions including mannitol or by any mechanical action, such as ultrasound-based approaches.

[0130] The vectors used herein may be formulated in any suitable vehicle for delivery. For instance, they may be placed into a pharmaceutically acceptable suspension, solution or emulsion. Suitable mediums include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[0131] Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. A colloidal dispersion system may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0132] The preferred doses and regimen may be determined by a physician, and depend on the age, sex, weight, of the subject, and the stage of the disease.

[0133] Host cells

[0134] A further aspect of the invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention.

[0135] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

[0136] The nucleic acids of the invention may be used to produce an antibody of the present invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cell"), and the like. The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present invention.

[0137] Pharmaceutical composition

[0138] A second object of the invention concerns a pharmaceutical composition for use in the treatment of Tauopathies in a subject in need thereof which comprises a therapeutically effective amount of a complex according to the invention.

[0139] According to the invention, the pharmaceutical composition of the invention can comprise the complex of the invention encoded by nucleic acids of the invention and administrated thanks to a vector comprising nucleic acids of the invention.

[0140] By a "therapeutically effective amount" is meant a sufficient amount of the complex of the invention to treat Tauopathies at a reasonable benefit / risk ratio applicable to any medical treatment.

[0141] It will be understood that the total daily dosage of the complexe and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific complex employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific complex employed; the duration and frequency of the treatment; drugs used in combination or coincidental with the specific complex employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the complex at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range per adult per day.

[0142] The presentation of the pharmaceutical compositions that contain the complex in any form according to the invention may be in any form that is suitable for subcutaneous, intraparenchymal, intracisternal, intrathecal, intraventricular, intramuscular, intravenous, intraocular, transdermal, topical, parenteral, rectal, intranasal and oral administration.

[0143] In the pharmaceutical compositions of the present invention for subcutaneous, intraparenchymal, intracisternal, intrathecal, intraventricular, intramuscular, intravenous, intraocular, transdermal, topical, parenteral, rectal, intranasal and oral administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.

[0144] Preferably, the pharmaceutical compositions contain vehicles that are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, which may include any potential relevant additives, permit the constitution of injectable solutions.

[0145] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0146] Solutions comprising the complex of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0147] The complex according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0148] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0149] Sterile injectable solutions are prepared by incorporating the active complex in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0150] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

[0151] Dose of the IL-2C or of the pharmaceutical composition of the invention

[0152] In another object, the IL-2C or pharmaceutical composition of the invention are administered to the subject in need thereof at doses ranged below 3.5 MIU / m2 / day (MIU = million international unit).

[0153] In a particular embodiment, the IL-2C or pharmaceutical composition of the invention may be administered under conditions that do activate and / or amplify Tregs without substantially activating and / or amplifying Teff, the skilled person may be able to adjust dosages to each patient and condition. Typically, the IL-2C or pharmaceutical composition of the invention are administered at a dose of about 0.05 to about 2 MIU / m2 / day, preferably 0.2 to about 1 MIU / m2 / day.

[0154] The amount of the IL-2C or pharmaceutical composition of the invention to administer thus preferably depends on the body surface area of the subject. The body surface area (BSA) is the measured or calculated surface of a human body. Various calculations have been published to determine the BSA without direct measurement. The Dubois & Dubois formula is commonly used in adults: BSA m2 = 0.007184 x weight kg 0.425 x height cm 0.725 = weight kg 0.425 x height cm 0.725 139.2 Another commonly used formula is the Mosteller formula adopted for use by the Pharmacy and Therapeutics Committee of the Cross Cancer Institute, Edmonton, Alberta, Canada: BSA m2 = weight kg X height cm 3600 = weight kg 0.5 X height cm 0.5 60.

[0155] Typically, the dosage according to the invention is below 3.5 MIU / day / subject, below 3.0 Million IU / day / subject, below 2.5 Million lU / day / subject, below 2.0 Million lU / day / subject, below 1.5 MUI / day / subject, below 1 MUI / day / subject, below 0.5 MUI / day / subject. The treatment is typically repeated, i.e. the above low doses of the IL-2C or pharmaceutical composition of the invention are administered several times to a subject, to progressively achieve the most substantial benefit. The dose and schedule of administration vary according to the deferment, preventive or therapeutic aim of the treatment, as well as to the disease to be treated / prevented / delayed. Treatment effect can be monitored by Treg measurements and dose and administration schedule adjusted accordingly.

[0156] Exemplary dosages are between 0.1 to 3 MIU, between 0.1 to 1.5 MIU, between 0.25 to 1 MIU. Preferred dosages are: 3.0 MIU / day / subject, 2.5 MIU / day / subject, 2.0 MIU / day / subject, 1.5 MIU / day / subject, 1.0 MIU / day / subject, 0.5 MIU / day / subject, 0.3 MIU / day / subject, 0.1 MIU / day / subject, 0.05 MIU / day / subject, 0.02 MIU / day / subject, 0.01 MIU / day / subject, or below 0.01 MIU / day / subject.

[0157] In one embodiment of the invention, the IL-2C or pharmaceutical composition of the invention are administered at a dose of about 0.05 to about 2 MIU / m2 / day, preferably 0.2 to about 1 MIU / m2 / day, or below 0.05 MIU / m2 / day.

[0158] In one embodiment of the invention, the IL-2C or pharmaceutical composition of the invention are administered from 1 to 7 days of daily administration, preferably between 3 to 5 days. Such treatment courses may be reproduced at different time periods, interrupted by periods with no treatment.

[0159] In one embodiment of the invention, the IL-2C or pharmaceutical composition of the invention are administered once per day during at least 3 consecutive days, preferably during 3 to 7, more preferably during 4 to 5 consecutive days, more preferably followed by a maintenance dose after 1 to 4 weeks, which may be repeated once or several times, each after 1 to 4 weeks.

[0160] In a preventive setting, the IL-2C or pharmaceutical composition of the invention may be administered at the above dosage in single shots, at different time intervals, e.g. once a week over long periods of time. Different protocols may be adjusted depending on the subject and disease. Maintenance dosage can be administered from two to eight weeks after the initiating cycle is completed. Preferably the maintenance dose is the same as the initiating dose. Combination and kit of part

[0161] In another aspect of the invention, the IL-2C or pharmaceutical composition of the invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an IL-2C of the invention and a further therapeutic active agent.

[0162] Particularly, the active agent(s) for treating a Tauopathy and / or Alzheimer's disorder include, but are not limited to: a cholinesterase inhibitor, such as donepezil, galantamine, rivastigmine and tacrine; a N-methyl D-aspartate (NMD A) antagonist, such as memantine; a BACE 1 inhibitor such as KMI-420, KMI-429, KMI- 358, KMI-370, TAK-070, and GSK188909, as reviewed, for example in Guo, T., and Hobbs, D.W., Curr. Med. Chem., vol. 13, no. 15, 2006, 1811-29; Evin, G., et al., Recent Pat CNS Drug Discov., vol. 6, no. 2, 2011, 91-106, and Citron, M., Nat Rev Drug Discov., vol.9, 2010, 387-398, which are hereby incorporated by reference and form part of the disclosure; y secretase inhibitors such as LY450139; passive anti-Ap immunotherapy in the form of monoclonal anti-Ap antibodies, such as but not limited to Aducanumab, Lecanemab, Donanemab, Bapineuzumab, Solanezumab, Gantenerumab; intravenous immunoglobulin; IV Ig (10%); intravenous Ig (IgG antibodies); Gammagard™ IV Ig (10%); PF-04360365; R1450; GSK933766A; active anti-Ap immunotherapy such as but not limited to ACC- 001, CAD-105, CAD-106, Affitope AD01, Affitope AD02, V950, UB311; non- steroidal anti-inflammatory drugs (NSAIDs); drugs enhancing Ap clearance such as but not limited to PF-04494700; drugs inhibiting Ap aggregation such as but not limited to ELND005, PBT2; drugs reducing Tau phosphorylation, such as but not limited to inhibitors of CDK5R1, MARK1, GSK3; passive anti-Tau immunotherapy in the form of monoclonal anti-Tau antibodies and variants thereof, including but not limited to nanobodies; active anti-Tau immunotherapy; APOE-related treatment approaches.

[0163] Another aspect of the present invention relates to i) an IL-2C or pharmaceutical composition of the invention, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of Tauopathies in a subject in need thereof.

[0164] As used herein, the term “simultaneous use” denotes the use of the IL-2C or pharmaceutical composition of the invention and at least one therapeutic active agent occurring at the same time. As used herein, the term “separate use” denotes the use of the IL-2C or pharmaceutical composition of the invention and at least one therapeutic active agent not occurring at the same time.

[0165] As used herein, the term “sequential use” denotes the use of the IL-2C or pharmaceutical composition of the invention and at least one therapeutic active agent occurring by following an order.

[0166] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0167] FIGURES:

[0168] Figure 1. IL-2C treatment induces a greater Treg amplification than “regular” low-dose IL-2 treatment in THY-Tau22 mice. A. Longitudinal assessment of Treg amplification in mice treated with IL-2 or IL-2C. B. Longitudinal assessment of the impact of IL-2 or IL-2C treatment on the amplification of effector T cells (Teff). The Fold Change was calculated relative to percentages in untreated, genotype-matched littermates. Schedule of the two types of treatment regimens is depicted below panel B. C. Assessment of Treg amplification 3 days after the last injection of the induction phase in mice treated with either IL-2 or IL-2C, i.e. when both treatments reach peak efficiency. D. Assessment of Teff amplification in IL-2- or IL-2C-treated mice at the peak of treatment efficiency. E. Assessment of Treg / Teff ratio in treated mice at the peak of treatment efficiency. F. Assessment of the impact of IL-2 or IL-2C treatment on the frequency of CD4+ effector memory T cells (TEM). G. Assessment of CD4+ Tregs / CD4+ TEM ratio in either IL-2- or IL-2C-treated mice.

[0169] Figure 2. Chronic IL-2C treatment restores long-term spatial memory in female THY-Tau22 mice. A. Mean total distance traveled by female mice on the first day of learning in the Barnes Maze test. B-C. Latency to find the correct hole for the first time in the Probe test at days 5 (B) and 12 (C). Statistical analyses were performed using an unpaired t-test or Mann- Whitney test (n=8-12 / group, *p<0.05, **p<0.01, ***p<0.001).

[0170] Figure 3. Chronic IL-2C treatment restores long-term spatial memory in male THY-Tau22 mice. A. Mean total distance traveled by male mice on the first day of learning in the Barnes Maze test. B-C. Percentage of visits to the correct target hole in the Probe test at day 5 (B) and 12 (C). Statistical analyses were performed using an unpaired t-test or Mann-Whitney test (n=10-17 / group, *p<0.05, **p<0.01, ***p<0.001).

[0171] Figure 4. Chronic IL-2C treatment modulates microglia and astrocytes reactivity in male THY-Tau22 mice. Quantification of disease-associated microglia markers (Al to A5), chemokines and Clqa (Bl to B4), and markers of reactive astrocytes (Cl to C3). Relative quantification was assessed by qPCR. Statistical analyses were performed using Mann-Whitney test (n=5-l 1 / group, *p<0.05, **p<0.01, ***p<0.001).

[0172] Figure 5. Chronic IL-2C treatment modulates astrocytes reactivity in female THY- Tau22 mice. A. Quantification of GFAP mRNA expression. Bl to B3. Quantification of the percentage of GFAP+ stained area by immunohistofluorescence. Cl to C3. Quantification of GFAP intensity by immunohistofluorescence. Statistical analyses were performed using Mann- Whitney test (n=5-6 / group, *p<0.05, **p<0.01, ***p<0.001).

[0173] EXAMPLE:

[0174] Material & Methods

[0175] Mice

[0176] THY-Tau22 mice (C57BL6 / J background were kindly provided by Luc Buee from Lille Neuroscience & Cognition Research Center (Inserm UMRS 1172). This transgenic murine model of AD-like Tau pathology was generated by overexpression of human four-repeat (1N4R) Tau mutated at sites G272V and P301S, under the control of the neuronal Thy 1.2 expression cassette. THY-Tau22 mice progressively develop hippocampal Tau deposition and innate neuroinflammation starting from 3 months of age, followed by cognitive deficits around 9 months. Non-transgenic wild-type (WT) littermates were used as controls.

[0177] Treatment

[0178] For Treg amplification, 3-month-old THY-Tau22 mice and WT littermates were injected intraperitoneally with 200 pL of either IL-2 / anti -IL-2 complex (IL-2C) or IgGl isotype control daily for 5 consecutive days for the induction phase, followed by a maintenance phase consisting in one injection every 3-4 days. The IL-2C was prepared by mixing 15 000 IU of IL- 2 (Proleukin) with 4.49 pg of mouse anti-human IL-2 (BD Bioscience, clone 5344.111) in phosphate buffer saline (PBS) IX. The complex was then incubated for 30min at 37°C before administration to mice.

[0179] Mouse Genotyping

[0180] Small fragments of the hindlimb digits or tails of the mice were harvested from newborn mice and digested in 81 pL digestion buffer and 14 pL of proteinase K (stock concentration 20 mg / mL) at 56°C for 3 hours or overnight, followed by 15 minutes at 95°C to inactivate the proteinase K. Samples were then diluted in 500 pL of distilled RNase-free water. 1 pL from each sample (or a Tau-positive control) was used for PCR amplification in a 0.2 mL PCR microtube along with a PCR mix containing 13.5 pL of RNase-free water, 5 pL of green Taq buffer, 0.5 pL of dNTPs (stock concentration 10 mM) , 2.5 pL of Tau23 bis primer, 2.5 pL of Tau25 primers (stock concentration 10 mM) and 0.35 pL of Taq polymerase (stock concentration 5 U / pL). PCR amplification of transgenic human Tau was carried out according to the following protocol: initial denaturation for 2min at 95°C then 40 cycles of denaturation (95°C; 30sec), annealing (55°C; 30sec) and elongation (72°C; 45sec). Amplified samples were then visualized by electrophoresis using a 2% agarose gel, allowing us to determine the presence or absence of target PCR product.

[0181] Behavior studies

[0182] Barnes Maze

[0183] The Barnes maze is a spatial learning and memory task based on the natural preference of mice for dark closed environments over bright open areas. The maze consists of an elevated flat circular platform with 20 circular holes located equidistantly at the periphery of the platform. A hidden escape box located under one of the target holes allows the mice to escape the platform. Fixed visual cues are displayed on distant walls around the maze for mice orientation. The test consisted in 4 days of learning, followed by one probe test on Day 5 with the escape box being removed, to be repeated on Day 12. Each mouse was given four trials per day during the learning phase and a single trial for each probe test. For each trial, mice were placed in an opaque starting box located in the center of the maze, ensuring a random starting orientation. After being released from the starting box, mice were allowed to freely explore the maze for a maximum of 300s (learning phase) or 120s (probe test). Indirect light and background noise was used as an incentive to motivate the mice to escape the maze. The trial was stopped once the mouse found the target hole or when the maximal duration of the trial was reached. Mice that did not find the escape box within 300s of the learning trial were gently pushed using a large beaker towards the target hole, into the escape box. Mice were left in the escape box for Imin before being returned to their home cage. The platform surface was carefully cleaned with 70% ethanol between each trial to remove any olfactory cues. An overhead video camera and automated video tracking software (Viewer3 BIOBSERVE) were used for recording and analyzing each behavior trial. Numbers of visits to the correct hole, speed, path length and escape latency to reach the target hole were monitored. Real-Time quantitative PCR

[0184] Brains of wild-type and THY-Tau22 mice previously treated with IL-2C or isotype control antibody were harvested at 9-9.5 months of age after transcardiac perfusion with PBS. Total RNA was extracted from the hippocampus using RNeasy lipid tissue mini kit (Qiagen). Following DNase treatment, RNA quality and quantity was measured using a Nanodrop 1000 (Thermo Fisher Scientific). For cDNA synthesis, 1 pg of total RNA was processed using Reverse Transcriptase SuperScript IV (Invitrogen). Relative expression of specific cDNAs was assessed by SYBR green-based real-time quantitative PCR using RT2 SYBR Green (Qiagen) and Light-Cycler 96 Instrument (Roche). Specificity of all primers was validated using Primer BLAST database analysis, and PCR efficiency (>90%) was validated after optimizing the concentration and annealing temperature of primers. Amplification conditions were: 95° for 5min, followed by 45 cycles at 95°C for 10s, then 15s at either 60°C or 56°C depending on the primer pairs, and 72° for 30s. A melting curve was generated at the end of the amplification cycles for assessing the specificity of the reaction. Peptidylprolyl isomerase A (PPIA) was used as a reference housekeeping gene for normalization. Relative expression was evaluated as fold changes using the mean of the WT + isotype control group as reference, and calculated as 2- AACt. All primers were ordered from Eurogentec (data not shown). The amplified genes were: PPIA, GFAP, C1QA, AMIG02, EMP1, TREM2, CCL4, P2RY12, ITGAX, CSF1R, CLEC7A, CCL3 and CCL5. Data were analyzed using LightCycler 96 software (Roche) and GraphPad Prism.

[0185] Immunohi stofluorescence

[0186] Mice were anaesthetized with ketamine (Imai gene®) and xylazine (Rompun™) in PBS, then transcardially perfused with PBS. Brains were harvested and hemi-brains post-fixed for 48 h at 4°C in 4% paraformaldehyde, then transferred overnight at 4°C in 30% sucrose / PBS solution and subsequently included in OCT. The cerebellum and olfactory bulb were removed and the midbrain was cut into 8 pm slices. To assess the extent of gliosis, brain slices were stained for GFAP (reactive astrocytes) and Ibal (activated microglia). First, the slices were hydrated for lOmin in TBS IX. After draining the TBS, 100 pL of blocking solution (TBS IX, 5% milk, 0.25% Triton XI 00) was added for 1 hour at room temperature to block non-specific binding sites and increase the signal-to-noise ratio. Sections were then incubated overnight at 4°C with rabbit anti-mouse Ibal (Wako, 1 / 200 dilution). The next day, sections were washed 4 times and incubated at room temperature with Alexa Fluor 488-conjugated anti-mouse GFAP (BD Biosciences, 1 / 200 dilution) and Alexa Fluor 555-conjugated donkey anti-rabbit IgG. After 4 washes, sections were stained with DAPI and coverslipped with ImmuMount medium. Image acquisition was carried out at 20X in fixed locations of the hippocampi, collecting 2 images of the dentate gyrus (DG), 2 images of the CAI region, 1 image of the CA2 region, and 1 image of the CA3 region. All images were acquired on an Olympus 1X81 inverted fluorescence microscope, using the same parameters of exposure and gain. Images were then analyzed and quantified using ImageJ software. To quantify gliosis across experimental groups, percentage of specifically-stained GFAP+ or Ibal+ area, as well as fluorescence intensity, was determined after standardized binarization of fluorescence images.

[0187] Flow cytometry analysis

[0188] Extent of peripheral Tregs amplification was monitored longitudinally in the blood. After blood harvesting (100 pL) in heparin, blood cells were resuspended in 90 pL PBS-0.5% Bovine Serum Albumin (BSA) for staining. For extracellular staining, cells were first incubated for lOmin at 4°C with 2 pL of Fc block (final concentration 12 pg / mL, mouse CD16 / CD32 blocker, clone 2.4G2). They were then incubated for 20min at 4°C with 10 pL of their respective extracellular antibody solution (BD Bioscience or Biolegends, dilution 1 / 200). Erythrocytes were lysed using 100 pL of BD FACS Lysis buffer. After washing, cells were permeabilized for intracellular staining using 100 pL of Foxp3 Fix / Perm buffer IX (Invitrogen) for 30min at 4°C, then washed with 100 pL of Foxp3 Perm / Wash buffer IX (Invitrogen). Cells were then incubated for lOmin at 4°C with 50 pL of Fc Block diluted in Foxp3 Perm / Wash Buffer IX (final concentration 60 pg / mL), before adding 50 pL of Foxp3 antibody solution (BD Bioscience; final concentration 1 pg / mL). Finally, cells were washed and resuspended in 300 pL of PBS-0.5% BSA and fluorescence data were collected using a Gallios flow cytometer (Beckman Coulter). For compensation settings, Ultra Compbeads (BD Biosciences) with corresponding antibodies were used. Results were analyzed using FlowJo Software (FlowJo LLC).

[0189] Results

[0190] IL-2C treatment induces heightened Treg amplification as compared to regular low dose IL-2 treatment

[0191] THY-Tau22 mice or WT littermates were treated for a total period of 21 days with either “regular” low dose IL-2 treatment or an optimized ultra-low dose IL-2 / anti-IL-2 complex (IL- 20) treatment. The IL-2C treatment regimen consisted in an induction phase of daily injections for 5 consecutive days, then one injection every 3 to 4 days for the maintenance phase. On the other hand, the regular low dose IL-2 treatment consisted in daily injections for 10 consecutive days. We assessed the peripheral amplification of regulatory T cells (Tregs; CD4+CD25+Foxp3+) and T effector cells (Teff; CD4+CD25+Foxp3-) in blood samples harvested bi-weekly over the treatment period. Whereas regular low-dose IL-2 treatment resulted in about 1.5-to-2-fold increase in the peripheral frequency of Tregs, in both WT and diseased mice, IL-2C induced a much greater amplification of Tregs, going up to a 4-to-5-fold increase in the frequency of peripheral Tregs compared to relevant control animals, i.e. either PBS- or isotype-treated genotype-matched littermates (Fig.lA). In parallel, we quantified the frequency of Teff and found that neither regular IL-2 nor IL-2C treatment significantly induced Teff amplification as compared to control -treated genotype-matched littermates, thereby confirming such IL-2 -based treatments specifically target and amplify Tregs (Fig.lB). Similar quantification at the peak of treatment efficiency confirmed the preferential and selective amplification of Tregs over Teff cells, as compared to control -treated genotype-matched littermates, and further suggests that IL-2C treatment resulted in heightened Tregs / Teff ratio as compared to “regular” low dose IL-2 treatment (Fig. 1C-E). Further characterization of CD4+ T subsets suggests that neither “regular” low dose IL-2 nor IL-2C treatment induce an increase in CD4+ effector memory T cells (TEM) as compared to control -treated genotype-matched littermates, thereby supporting that the beneficial impact of such treatments is independent of TEM cells (Fig. IF). In addition, IL-2C treatment resulted in heightened Tregs / Ti i ratio as compared to “regular” low dose IL-2 treatment, although this effect does not reach significance in THY-Tau22 mice (Fig. 1G). Altogether, these data evidence the heightened efficiency of IL- 2C treatment, as compared to the regular low dose IL-2 approach, to selectively amplify Tregs in both healthy conditions and in the context of AD-like Tau pathology.

[0192] Peripheral amplification of Tregs via IL-2C treatment delays the onset of cognitive deficits in THY-Tau22 mice

[0193] THY-Tau22 mice or WT littermates were treated chronically with IL-2C from 3 months of age and until behavior assessment, starting with daily injections for 5-days (induction phase) followed by one injection every 3-4 days (maintenance phase). Males were assessed for cognitive deficits at 8.5-9 months, while females were assessed at 9-9.5 months, as disease development is slightly faster in males than females THY-Tau22 mice. Short- and long-term spatial memory were assessed using the Y-maze and Barnes maze test, respectively.

[0194] We sought to assess long-term memory and cognitive deficits using the Barnes Maze, during which mice are taught to use surrounding visual cues to find a target hole towards an escape box. We first analyzed the mice’s path length on the first day of learning to assess any potential impact of genotype on spontaneous exploratory behavior. As THY-Tau22 females do not exhibit differences in spontaneous exploratory behavior compared to WT littermates (Fig. 2A), we analyzed the primary latency, i.e. the time spent to find the target hole for the first time, as the main read out to assess cognitive deficits in the two probe tests. On the probe test at day 5, isotype-treated THY-Tau22 mice displayed significantly higher primary latency than WT littermates, thus evidencing strong cognitive deficits in THY-tau22 mice. In contrast, IL-2C- treated THY-Tau22 mice display similar primary latency as WT mice, either isotype- or IL-2C- treated, supporting the recovery of cognitive functions upon IL-2C treatment (Fig. 2B). A similar trend was observed when repeating the probe test on day 12, although the data could not reach significance (Fig.2C).

[0195] Similar analyses were conducted on male mice. In contrast to females, males THY- Tau22 exhibited significant differences in their spontaneous exploratory behavior as compared to WT littermates, as highlighted by the significantly higher path length on the first day of learning (Fig. 3A). To avoid any potential bias in the analysis, in males we thus used the percentage of visits to the correct target hole as a standardized measure for assessing the impact of treatment on memory deficits. Whereas isotype-treated THY-Tau22 mice display significantly lower percentages of correct visits than WT littermates, IL-2C-treated THY-Tau22 males display similar percentages as isotype-treated WT mice, on both probe tests at day 5 and 12 (Fig. 3B-C). These results further support the efficacy of IL-2C treatment at restoring longterm memory and cognitive functions associated with Tau-pathology in both males and females.

[0196] Amplification of Tregs via IL-2C treatment modulates Tau-associated neuroinflammatory profiles in THY-Tau22 mice

[0197] The development of Tau pathology is associated with a chronic innate neuroinflammatory response mediated by microglia and astrocytes. We first assessed the impact of IL-2C treatment on the expression of typical neuroinflammatory markers using RT- qPCR.

[0198] Disease-Associated Microglia (DAM) are a hallmark of the neuroinflammatory response associated with Tau pathology. Whereas a strong upregulation of DAM markers is observed in control -treated THY-Tau22 compared to WT mice, IL-2C treatment significantly decreases the expression of such DAM markers in THY-Tau22 mice. (Fig. 4A1 to 4A3). The most striking examples are for ITGAX and CLEC7A, which are significantly decreased in IL- 2C-treated as compared to IgG-treated THY-Tau22 mice. A similar trend is observed for the DAM marker TREM2. Whereas it is significantly upregulated in the brain of control -treated THY-Tau22 compared to WT mice, no statistical difference is observed between IL-2C-treated THY-Tau22 and WT mice, suggesting that IL-2C treatment tends to also normalize the expression of TREM2 (Fig. 4A1 to 4A3). In the same line, a similar trend is observed for the T-cell-attracting chemokines CCL3, CCL4 and CCL5, as well as the pro-inflammatory complement protein Clqa, which are strongly upregulated in the brain of isotype-treated THY- Tau22 compared to WT mice. Conversely, IL-2C treatment significantly dampens the expression of CCL4 and tends to decrease CCL3, CCL5 and Clqa in the brain of THY-Tau22 mice, which are no longer statistically different from their WT littermates (Fig. 4B1 to 4B4). Besides, the expression of markers from homeostatic microglia, such as P2RY12 and CSF1R, is downregulated in the brain of IL-2C- vs isotype-treated THY-Tau22 mice, further supporting an altered pattern of microglial profiles upon IL-2C treatment (Fig. 4A4 & 4A5). Altogether, these data suggest that IL-2C treatment contributes to reshape the functional profiles of microglia associated with Tau pathology, including decreased DAM markers and a potentially rebalanced pattern of homeostatic microglia.

[0199] Secreted Clqa is known to contribute modulating the functional profile of astrocytes, together with TNFa and IL- la, by promoting their functional differentiation towards pro- inflammatory subsets. We thus quantified the expression of reactive astrocytes’ markers in male mice. Whereas only a tendency towards GFAP upregulation is observed in isotype-treated THY-Tau22 compared to WT mice, IL-2C treatment increases GFAP expression selectively in THY-Tau22 but not WT mice, supporting that IL-2C promotes global astrocytes reactivity in the context of Tau pathology (Fig. 4C1). We further assessed the expression of markers for given astrocyte subsets, such as AMIG02 for pro-inflammatory astrocytes and EMP1 for neurotrophic astrocytes subsets. Whereas AMIG02 is close to be significantly upregulated in isotype-treated THY-Tau22 compared to WT mice, IL-2C treatment tends to decrease AMIG02 expression selectively in THY-Tau22 mice (Fig. 4C2). Similarly, IL-2C treatment also tends to decrease EMP1 expression preferentially in THY-Tau22 mice (Fig. 4C3). Overall, these data suggest that IL-2C treatment modulates astrocytes reactivity associated with Tau pathology, possibly by altering the balance of reactive astrocytes subsets.

[0200] Chronic IL-2C treatment also seems to modulate global astrocyte reactivity in female mice. Hence, isotype-treated THY-Tau22 female mice exhibit a marked upregulation of GFAP compared to WT littermates, which significance is lost upon treatment with IL-2C. This effect can be observed both at the transcriptomic level (Fig. 5A) and by immunohistofluorescence analysis (Fig. 5B1 to B3; Fig 5C1 to 5C3), further supporting the capacity of chronic IL-2C treatment at modulating astrocytes reactivity associated with Tau pathology.

[0201] REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0202] Schlepckow et al,. Stimulation of TREM2 with agonistic antibodies — an emerging therapeutic option for Alzheimer’ s disease. Lancet Neurology 2023.

[0203] Carling et al,. Friend turned foe: TREM2 agonist in battles against tau. J Exp Med 2022 - Editorial on Jain et al, J Exp Med 2022.

[0204] Rosenzweig et al,. PD-1 / PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model. Nature Comm 2019.

Claims

CLAIMS:

1. An IL-2 / anti-IL-2 complex (IL-2C) for use in the treatment of Tauopathies.

2. An IL-2C for use according to the claim 1 wherein the IL-2C acts as a booster of Tregs cells.

3. An IL-2C for use according to the claims 1 or 2 wherein Tauopathies include but are not limited to primary Tauopathies (FTLD-tau) like progressive supranuclear palsy (PSP), Pick's disease, corticobasal degeneration (CBD), argyrophilic grain disease, globular glial tauopathy, FTLD-MAPT (FTLD with genetic mutations in the gene encoding tau), behavioural variant of Frontotemporal dementia, progressive non-fluent aphasia, semantic dementia, as well as secondary tauopathy like Alzheimer's Disease.

4. An IL-2C for use according to the claim 3 wherein the Tauopathy is Alzheimer's Disease.

5. An IL-2C for use according to the claims 1 to 4 wherein the IL-2 is the human IL-2.

6. A method of treating Tauopathies in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of an IL-2 / anti-IL-2 complex (IL-2C).

7. A pharmaceutical composition for use in the treatment of Tauopathies in a subject in need thereof which comprises a therapeutically effective amount of an IL-2 / anti-IL-2 complex (IL-2C).

8. An IL-2C according to the claims 1 to 5 or a pharmaceutical composition according to the claim 7 wherein the IL-2C or the pharmaceutical composition are administered to the subject in need thereof at doses ranged below 0.01 MIU / day / subject.

9. An i) IL-2 / anti-IL-2 complex or pharmaceutical composition according to the claim 7, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of Tauopathies in a subject in need thereof.

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