Reducing systemic regulatory t cell levels or activity for treatment of disease and injury of the CNS

Abstract

To provide a pharmaceutical composition comprising an active agent that causes reduction of the level of systemic immunosuppression in an individual for use in treating a disease, disorder, condition or injury of the CNS that does not include the autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RRMS).SOLUTION: A pharmaceutical composition according to the present invention is intended for administration by a dosage regimen comprising at least two courses of therapy, each course of therapy comprising in sequence a treatment session followed by an interval session.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present invention generally relates to methods and compositions for treating diseases, disorders, conditions or injuries of the central nervous system (CNS) by temporarily reducing the level of systemic immunosuppression in the circulation.

Background Art

[0002] Most lesions of the central nervous system (CNS) are part of disease progression and are mediated through a common neuroinflammatory component that is a cause of disease aversion. One of these lesions is Alzheimer's disease (AD), i.e., the accumulation of amyloid-β (Aβ) peptide aggregates has been suggested to play an important role in the inflammatory cascade within the CNS and ultimately cause neuronal damage and tissue destruction, an age-related neurodegenerative disease characterized by the progressive loss of memory and cognitive functions (Akiyama et al., 2000; Hardy & Selkoe, 2002; Vom Berg et al., 2012). Despite the long-term neuroinflammatory responses in various neurodegenerative diseases, over the past decade, clinical and preclinical studies have questioned why anti-inflammatory drugs are lacking while immunosuppression-based therapies are being investigated in neurodegenerative diseases (Breitner et al., 2009; Group et al., 2007; Wyss-Coray & Rogers, 2012). The inventors provide a novel answer to overcome the drawbacks of existing treatments for AD and similar diseases and injuries of the CNS; this method is based on the inventors' unique understanding of the roles of various components of the systemic and central immune systems in the maintenance and repair of the CNS.

Summary of the Invention

[0003] In one aspect, the present invention provides a pharmaceutical composition for use in treating an autoimmune neuroinflammatory disease, a CNS disease, disorder, condition or injury other than relapsing-remitting multiple sclerosis (RRMS), which comprises an active agent that causes a reduction in the level of systemic immunosuppression in an individual, the pharmaceutical composition being for administration according to a dosing schedule comprising at least two treatment courses, each treatment course in turn comprising a treatment period followed by a non-treatment interval.

[0004] In another aspect, the present invention provides a method for treating a central nervous system (CNS) disease, disorder, condition or injury other than relapsing-remitting multiple sclerosis (RRMS) of an autoimmune neuroinflammatory disease, the method comprising administering to an individual in need thereof a pharmaceutical composition according to any one of claims 1 to 24, the pharmaceutical composition being administered according to a dosing schedule comprising at least two treatment courses, each treatment course in turn comprising a treatment period followed by a non-treatment interval. BRIEF DESCRIPTION OF THE DRAWINGS

[0005]

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Mode for Carrying Out the Invention

[0006] Foxp3 in a mouse model of Alzheimer's disease (AD-Tg mice) + Short-term transient depletion of regulatory T cells (Tregs) results in improved mobilization of leukocytes into the CNS via the choroid plexus of the brain, increased numbers of anti-inflammatory monocyte-derived macrophages mo-MΦ and CD4 + T cells, as well as increased Foxp3 accumulation in the brain + A marked enrichment of Tregs has been found according to the present invention. Moreover, the long-term effects of just a single treatment period cause a reduction in hippocampal gliosis and reduced mRNA expression levels of pro-inflammatory cytokines in the brain. Importantly, the effects on disease pathology include reduced brain amyloid-beta (Aβ) plaque burden in the two brain regions that exhibit robust Aβ plaque pathology in AD-Tg mice, namely the hippocampal dentate gyrus and the cerebral cortex (layer 5). Most importantly, short-term transient depletion of Tregs results in dramatic improvements in spatial learning and memory, thereby achieving a cognitive performance similar to that of wild-type mice (Examples 2 and 3). In summary, from these findings, short-term Treg depletion, when followed by a period of non-intervention, causes a temporary interruption of Treg-mediated systemic immunosuppression in AD-Tg mice, which enables the recruitment of cells that resolve inflammation (mo-MΦ and Tregs) to the brain and results in the dissipation of the neuroinflammatory response, the elimination of Aβ, and the restoration of cognitive decline. These findings strongly contradict the common sense in this research field, according to which an increasing systemic immune response would cause a mitigation of the neuroinflammatory response and is thus contrary thereto. Instead, the findings of the present inventors show that enhancing the systemic response by a short-term, brief and transient decrease in systemic Treg-mediated suppression is necessary to achieve the accumulation of inflammation-resolving immune cells, including Tregs themselves, in the brain and thus to combat AD pathology

[0007] The specificity of the inventors' efforts shown in this specification has been demonstrated by using several independent experimental frameworks, as detailed below. Briefly, the inventors first used immunomodulatory compounds in two different dosing regimens that had opposite effects on peripheral Treg levels, on CP activation, and on disease pathology, namely, a daily dosing regimen that enhanced peripheral Treg levels (Weber et al., 2007) and a weekly dosing regimen that was found to reduce peripheral Treg levels (Examples 3 and 5). The inventors have also provided a direct functional link between peripheral Treg levels and disease pathology, where the inventors have shown in AD-Tg mice that either by transient in vivo genetic depletion of Tregs (Example 2) or by pharmacological inhibition of Foxp3 function in AD-Tg mice (Examples 3 and 4), these manipulations result in activation of CP to promote leukocyte transport to the CNS, accumulation of inflammatory resolving immune cells at the lesion site, elimination of brain Aβ plaques, and shifting of the immunological environment of the brain parenchymal tissue towards inflammatory resolution.

[0008] Furthermore, when the copolymer-1 of the universal antigen is administered rarely over a limited period (representing one treatment period), Treg-mediated systemic immunosuppression is reduced, and the selective infiltration of leukocytes into the CNS is improved by the increased activity of the choroid plexus entry of the brain, thereby resulting in a dramatic beneficial effect on Alzheimer's disease pathology (Example 3). On the other hand, when copolymer-1 is administered daily, Treg immunosuppression is enhanced (Hong et al., 2005; Weber et al., 2007), but no beneficial effect on the disease pathology is shown, or rather, some minor adverse effects are shown (Example 5), as found according to the present invention. The inventors of the present invention further show herein that by inhibiting p300 with a specific small molecule inhibitor (p300i) or by inhibiting the interaction with the PD-1 receptor with an anti-PD-1 antibody, either way, directly interfering with the Treg activity of Foxp3 improves the choroid plexus entry activity in AD-Tg mice and alleviates the Alzheimer's disease pathology (Example 4).

[0009] Importantly, each of these examples provided by the inventors reveals different interventions that cause a short-term decline in systemic immunosuppression: Copolymer-1 acts as an immunomodulatory compound, p300i acts as a small molecule that reduces Foxp3 acetylation and Treg function, and anti-PD-1 is used as a neutralizing antibody against PD-1 expressed on the surface of Tregs. These therapeutic approaches were used for a short treatment period that mainly increased peripheral IFN-γ levels and IFN-γ-producing cells, thus activating the brain choroid plexus, which in turn temporarily enhanced the immune response peripherally by enabling selective infiltration of T cells and monocytes into the CNS and homing of these cells to the lesion and neuroinflammatory sites. Repeated treatment periods interrupted by non-treatment intervals were also found to dramatically improve treatment efficacy compared to just one treatment period (Example 4). The subsequent time intervals without treatment promoted a temporary enhancement in Treg levels and Treg activity in the brain, dissipation of neuroinflammation, and induced environmental conditions favorable for CNS healing and repair, subsequently enabling tissue recovery. In each of these cases, the effect on brain pathology was robust, accompanied by dissipation of the neuroinflammatory response, elimination of amyloid-beta plaques from the brains of AD mice, and recovery of cognitive decline. The specificity of the current approach is further demonstrated in a transgenic mouse model of AD using a genetic model of transient depletion of regulatory T cells (Example 2). + It has been demonstrated in a transgenic mouse model of AD using a genetic model of transient depletion of regulatory T cells (Example 2).

[0010] Thus, systemic Foxp3 + CD4 +It has been found according to the present invention that Treg-mediated immunosuppression acts at least in part by inhibiting the IFN-γ-dependent activation of CP, which is necessary to organize the recruitment of inflammatory resolving leukocytes to the CNS; Schwartz & Baruch, 2014b), thereby preventing the ability to repel AD pathology. Systemic Tregs are very important for maintaining autoimmune homeostasis and protecting from autoimmune diseases (Kim et al., 2007). However, the inventors' findings suggest that under neurodegenerative conditions, the ability to initiate this response is hampered by systemic Tregs when a repair immune response is needed in the brain. Nevertheless, according to the inventors' results, Tregs are required in the brain, home to the site of the neuropathology, and locally achieve anti-inflammatory activity. The present invention represents a unique and unexpected solution to the apparently opposing needs in repelling progressive neuron death, as in the case of AD: namely, temporarily reducing / inhibiting Tregs in circulation instead of increasing Tregs in the diseased brain. Thus, a short-term and temporary decrease in peripheral immunosuppression enables the recruitment of anti-inflammatory cells (including Tregs and mo-MΦ) to the plaque sites in the brain while causing long-term effects on the pathology. Notably, however, a temporary decrease in systemic Treg levels and Treg activity may contribute to disease remission via additional mechanisms, including assisting a CNS-specific protective autoimmune response (Schwartz & Baruch, 2014a), or enhancing the levels of circulating monocytes that play a role in the clearance of vascular Aβ (Michaud et al., 2013).

[0011] Despite the fact that various neurodegenerative diseases with different etiologies share common local neuroinflammatory components, our results strongly argue against simplifying and characterizing all CNS pathologies as diseases that would uniformly benefit from systemic anti-inflammatory treatment. Thus, autoimmune inflammatory brain lesions, such as relapsing-remitting multiple sclerosis (RRMS), benefit from continuous systemic administration of anti-inflammatory and immunosuppressive drugs to achieve long-term peripheral immunosuppression, but long-term peripheral immunosuppression is either ineffective or detrimental to the pathology in chronic neurodegenerative diseases, such as in the case of AD, primary progressive multiple sclerosis (PP-MS), and secondary progressive multiple sclerosis (SP-MS) (Example 5). Moreover, our findings have helped to clarify misunderstandings regarding the role of systemic Tregs versus tissue-associated Tregs in these pathologies (He & Balling, 2013). Since the immune-brain axis is part of lifelong brain plasticity (Baruch et al., 2014) and various neurodegenerative diseases are mainly age-related, our findings also imply a more general phenomenon where systemic immunosuppression impairs brain function. Thus, short-term cyclic processes that reduce systemic immunosuppression may represent therapeutic or even preventive approaches that are applicable to a wide range of brain pathologies, including AD and age-related dementia.

[0012] Importantly, our approaches and findings herein in the AD mouse model do not directly target any disease-specific factors in AD, such as amyloid-beta or tau pathology, etc., but rather reveal a novel approach that is expected to be clinically applicable in a wide range of CNS pathologies, namely, temporarily reducing systemic Treg-mediated immunosuppression to enhance the recruitment of inflammatory-resolving immune cells to the lesion sites within the CNS.

[0013] Considering the unexpected results described above, the present invention provides a pharmaceutical composition comprising an active agent that causes a reduction in the level of systemic immunosuppression in an individual for use in treating autoimmune neuroinflammatory diseases, CNS diseases, disorders, conditions or injuries excluding relapsing-remitting multiple sclerosis (RRMS), the pharmaceutical composition being for administration according to a dosing schedule comprising at least two treatment cycles (where each treatment cycle in turn comprises a treatment period followed by a non-treatment interval).

[0014] In certain embodiments, the dosing schedule is adjusted such that the level of systemic immunosuppression is temporarily reduced.

[0015] The term "treating" as used herein refers to means for obtaining a desired physiological effect. This effect may be therapeutic in terms of partially or completely curing the disease and / or symptoms believed to be caused by the disease. The term refers to inhibiting the disease, i.e., arresting or retarding its development, or to ameliorating the disease, i.e., causing regression of the disease.

[0016] The term "non-treatment period" is used interchangeably herein with the term "period of non-treatment" and refers to a period during which no active agent is administered to an individual undergoing treatment.

[0017] The term "systemic presence" of regulatory T cells as used herein refers to the presence of regulatory T cells (as measured by their level or activity) in the circulating immune system (i.e., blood, spleen and lymph nodes). It is a well-known fact in the field of immunology that the cellular population profile in the spleen is reflected in the cellular population profile in the blood (Zhao et al., 2007).

[0018] The treatment of the present invention is applicable both to patients showing an increase in systemic immunosuppression and also to patients not showing such an increase. Sometimes, the individual in need of treatment according to the present invention has a certain level of peripheral immunosuppression, in which case such a level of peripheral immunosuppression is reflected by an increased frequency or number of Tregs in the circulation, and / or their enhanced functional activity, and / or a decrease in IFNγ-producing leukocytes, and / or a decreased proliferation of leukocytes in response to stimulation. The increase in the frequency or number of Tregs can be in the total number or as a proportion of the total CD4 cells. For example, an animal model of Alzheimer's disease has been found according to the present invention to have a greater frequency of Foxp3 in CD4 cells when compared to wild-type mice. However, even if in said individual the levels of systemic Treg cells do not increase, their functional activity does not increase, the levels of IFNγ-producing leukocytes do not decrease, or the proliferation of leukocytes in response to stimulation does not decrease, the method of the present invention for reducing the level or activity of systemic immunosuppression is effective in treating CNS diseases, disorders, conditions or injuries other than RRMS of autoimmune neuroinflammatory diseases. Importantly, said systemic immunosuppression may also involve additional immune cell types other than Tregs, such as myeloid-derived suppressor cells (MDSC) (Gabrilovich & Nagaraj, 2009).

[0019] The level of systemic immunosuppression may be detected by a variety of methods well known to those skilled in the art. For example, the level of Tregs may be measured by flow cytometry analysis of peripheral blood mononuclear cells or T lymphocytes immunostained for either a cell surface marker or an intracellular marker of the nucleus of Tregs (Chen & Oppenheim, 2011), the CD45 marker of lymphocytes, the TCR-β marker, or the CD4 marker, or by measuring the amount of antibody that specifically binds to the cells. The functional activity of Tregs may be measured by various assays; for example, the thymidine incorporation assay is commonly used, in which case CD4 + CD25 - The suppression of the proliferation of T cells (conventional T cells) stimulated with anti-C D3 mAb is 3 measured by [3H] thymidine incorporation or by using CFSE (5-(and 6)-carboxyfluorescein diacetate succinimidyl ester, which can enter cells; cell division is measured as the successive halving of the fluorescence intensity of CFSE). The number or activity or proliferative capacity of IFNγ-producing leukocytes can be readily evaluated by those skilled in the art using methods known in the art; for example, the level of IFNγ-producing leukocytes is measured by flow cytometry analysis of peripheral blood mononuclear cells after short-term ex vivo stimulation and Golgi stop, and by immunostaining with intracellular staining of IFNγ (for example, using the BD Biosciences Cytofix / cytoperm™ fixation / permeabilization kit), or by collecting the conditioned medium of these cells and quantifying the level of secreted cytokines using ELISA, or by comparing the ratios of various cytokines in the conditioned medium (for example, IL2 / IL10, IL2 / IL4, INFγ / TGFβ, etc.). The level of MDSCs in human peripheral blood is, as described (Kotsakis et al., 2012), for example, DR - / LIN - / CD11b+ cells, DR - / LIN - / CD15+ cells, DR - / LIN - It can be readily evaluated by those skilled in the art by using flow cytometry analysis of the frequencies of / CD33+ cells and DR(- / low) / CD14+ cells.

[0020] In humans, peripheral / systemic immunosuppression may be considered to be elevated when: the total number of Tregs in circulation is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more greater than that in a healthy control population; the proportion of Treg cells in the total CD4+ cells is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more higher than that in a healthy control population; or the functional activity of Tregs is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more higher than that in a healthy control population. Alternatively, peripheral / systemic immunosuppression may be considered to be elevated when: the level or activity of IFNγ-producing leukocytes is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower than the level or activity in a healthy control population; or the proliferation of leukocytes in response to stimulation is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower than the proliferation in a healthy control population.

[0021] An agent may be considered to be an agent that causes a decrease in the level of systemic immunosuppression when: when the agent is administered to an individual, the total number of Tregs in the circulation of this individual decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level before administration of the agent; the proportion of Treg cells in the total CD4+ cells decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the proportion in a healthy control population; or the functional activity of Tregs decreases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the level before administration of the agent. Alternatively, an agent may be considered to be an agent that causes a decrease in the level of systemic immunosuppression when: when the agent is administered to an individual, the total number or the activity of IFNγ-producing leukocytes increases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%; or the proliferation of leukocytes in response to a stimulus increases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the proliferation in a healthy control population.

[0022] The agent used according to the present invention may be any agent that downregulates the level or activity of regulatory T cells or inhibits their activity. However, alternatively, even if not known for use according to the treatment process according to the present invention, such agents may be limited to the group of such agents excluding agents selected from the group consisting of (i) dopamine or a pharmaceutically acceptable salt thereof, (ii) a dopamine precursor or a pharmaceutically acceptable salt thereof, (iii) an agonist of the dopamine receptor type 1 family (D1-R agonist) or a pharmaceutically acceptable salt thereof, and (iv) an antagonist of the dopamine receptor type 2 family (D2-R antagonist) or a pharmaceutically acceptable salt thereof.

[0023] In certain embodiments, the treatment period includes administering the pharmaceutical composition to the individual, and the treatment period is maintained until at least the level is lower than the reference standard, and administration is suspended during the period of the intermittent phase, and the intermittent phase is maintained as long as the level is lower than the reference standard. The reference standard may be selected from (a) the level of the systemic presence or activity of regulatory T cells or myeloid-derived suppressor cells measured in the most recent blood sample obtained from the individual prior to said administration, or (b) the level of the systemic presence or activity of regulatory T cells or myeloid-derived suppressor cells characteristic of a population of individuals suffering from a CNS disease, disorder, condition or injury.

[0024] Alternatively, the treatment period includes administering the pharmaceutical composition to the individual, and the treatment period is maintained until at least the systemic presence or level of IFNγ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation increases beyond the reference standard, and administration is suspended during the period of the intermittent phase, and the intermittent phase is maintained as long as said level exceeds the reference standard, provided that in this case the reference standard is selected from (a) the level of the systemic presence or activity of IFNγ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation measured in the most recent blood sample obtained from the individual prior to said administration, or (b) the level of the systemic presence or activity of IFNγ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation characteristic of a population of individuals suffering from a CNS disease, disorder, condition or injury.

[0025] The lengths of the treatment period and the intermittent phase are determined by a physician in a clinical trial directed to a particular patient population without the need to monitor the level of immunosuppression on an individual basis and may then be consistently applied to this patient population.

[0026] In certain embodiments, the treatment period may be between 3 days and 4 weeks in length, for example, between 1 week and 4 weeks in length.

[0027] In certain embodiments, the intermittent period may be between 1 week and 6 months, for example, may be of a length between 2 weeks and 6 months, particularly of a length between 3 weeks and 6 months.

[0028] During the treatment period, the administration of the pharmaceutical composition may be repeated administration. For example, the pharmaceutical composition may be administered once daily, or once every 2 days, 3 days, 4 days, 5 days or 6 days, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks. These frequencies may be applicable to any active agent, may be based on common practice generally used in this technical field, and may ultimately be determined by a physician in clinical trials. Alternatively, the frequency of repeated administration during the treatment period may be adapted according to the nature of the active agent. However, in this case, for example, small molecules may be administered daily; antibodies may be administered once every 3 days; and copolymer 1 may be administered once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks. When the agent (such as copolymer 1, etc.) is administered at a relatively low frequency during the treatment period, for example, once a week during a 1-month treatment period, or once a month during a 6-month treatment period it must be understood that after this treatment period, a non-treatment intermittent period follows, the length of which is longer than the period between repeated administrations during the treatment period (i.e., in this example, longer than 1 week or 1 month respectively). The 1-week or 1-month break between administrations during the treatment period in this example is not considered an intermittent period.

[0029] The lengths of the treatment period and the intermittent period may be adjusted according to the administration frequency, such that, for example, a frequency of administering the active agent once every 3 days may result in a treatment period of 6 days or 9 days and an intermittent period starting accordingly.

[0030] As an alternative to a pre-determined general treatment therapy, the level of immunosuppression is individually monitored for the level or activity of Treg cells (or IFN-γ-producing leukocytes) (or the rate of leukocyte proliferation in response to stimulation), and the treatment period, dosing frequency, and intermission period are empirically and personally adjusted so as to be determined from the results of the monitoring, such that it may be adjusted to the desired level for each patient being treated (personalized medicine).

[0031] Accordingly, the length of the treatment period is determined by (a) monitoring the level of the systemic presence or activity of regulatory T cells in an individual by measuring the level in a blood sample obtained from the individual within a predetermined period after said administration, (b) comparing the level measured in (a) with the above-mentioned reference criteria to clarify whether the level is different from the reference criteria, (c) determining, based on the relationship of the level measured in (a) to the reference criteria, whether to continue the treatment period by repeating the administration or to start the next intermission period by stopping the repetition of the administration, and (d) repeating the administration or starting the next intermission period as determined in (c). Alternatively, the level of IFN-γ-producing leukocytes, or the rate of leukocyte proliferation in response to stimulation, may be monitored and compared with appropriate reference criteria as described above.

[0032] Similarly, the length of the intermittent period is monitored by: (a) measuring the level of the systemic presence or activity of regulatory T cells in an individual in a blood sample obtained from the individual within a predetermined period after said administration; (b) comparing the level measured in (a) with the above-mentioned reference standard to clarify whether the level is different from the reference standard; (c) based on the relationship between the level measured in (a) and the reference standard, determining whether to initiate a new treatment course by repeating the administration and steps (a) and (b), or whether to extend the intermittent period by repeating only steps (a) and (b); and (d) according to the determination in (c), repeating the administration and steps (a) and (b), or only steps (a) and (b) as the case may be. Alternatively, the level of IFNγ-producing leukocytes or the proliferation rate of leukocytes in response to stimulation may be monitored and compared with appropriate reference standards as described above.

[0033] In any case, the dosing schedule, i.e., the lengths of the treatment period and the intermittent period, are adjusted such that, for example, a decrease in the level of immunosuppression as measured by a decrease in the level of the systemic presence or activity of regulatory T cells in an individual is transient.

[0034] In certain embodiments, the above-mentioned predetermined period, i.e., the time elapsed between the most recent administration of the active agent and the monitoring step, is between 2 days and 6 months.

[0035] In certain embodiments, the regulatory T cells to be monitored are FoxP3+ cells that express one or more of CD25, CD127, GITR, CTLA-4 or PD-1, or CD4+ cells selected from FoxP3+ cells that express one or more of the surface molecules CD25, CD127, GITR, CTLA-4 or PD-1. Specifically, a common phenotype of regulatory T cells is CD4 + cells, or, CD4+ cells selected from FoxP3+ cells that express one or more of the surface molecules CD25, CD127, GITR, CTLA-4 or PD-1. Specifically, a common phenotype of regulatory T cells is CD4 cells, or, CD4+ cells selected from FoxP3+ cells that express one or more of the surface molecules CD25, CD127, GITR, CTLA-4 or PD-1. Specifically, a common phenotype of regulatory T cells is CD4 - cells.+ CD25 + FoxP3 + cells or CD4 + CD25 + FoxP3 - cells.

[0036] A variety of agents that can reduce the levels of regulatory T cells are known in the art (Colombo & Piconese, 2007), and these agents can be used in accordance with the present invention. Each of the cited publications below is incorporated by reference as if fully disclosed herein.

[0037] Accordingly, the agent may be selected from the following, but the agent is not necessarily limited thereto: (i) antibodies, such as (a) anti-PD-1, (b) anti-PD-L1, (c) anti-PD-L2 (Coyne & Gulley, 2014; Duraiswamy et al., 2014; Zeng et al., 2013), (d) anti-CTLA-4 (Simpson et al., 2013; Terme et al., 2012), (e) anti-PD-1 in combination with interferon α (Terawaki et al., 2011), (f) anti-PD-1 in combination with anti-CTLA-4, (g) anti-CD47 (Tseng et al., 2013), (h) anti-OX40 (Voo et al., 2013), (i) anti-VEGF-A (bevacizumab) (Terme et al., 2013), (j) anti-CD25 (Zhou et al., 2013), (k) anti-GITR (GITR-activating mAb (DTA-1) (Colombo & Piconese, 2007), (l) anti-CCR4, (m) anti-TIM-3 / galectin 9 (Ju et al., 2014), (n) anti-killer cell immunoglobulin-like receptor (KIR), (o) anti-LAG-3 or (p) anti-4-1BB; (ii) any combination of (a) to (p); (iii) any combination of (a) to (p) in combination with an adjuvant, such as anti-CTLA-4 antibody in combination with anti-OX40 antibody and TLR9 ligand (e.g., CpG, etc.) (Marabelle et al., 2013); (iv) small molecules selected from the following: (a) p300 inhibitor (Liu et al., 2013), such as gemcitabine (low dose) (Shevchenko et al., 2013), or C646 or an analog thereof, i.e., a compound of formula I below:

Chemical formula

[0038] In certain embodiments, the agent is an anti-PD-1 antibody, i.e., an antibody specific for PD-1.

[0039] Many anti-PD-1 antibodies are known in the art. For example, the anti-PD-1 antibody used in accordance with the present invention may be selected from the anti-PD-1 antibodies disclosed by Ohaegbulam et al. (Ohaegbulam et al., 2015; the entire content of which is incorporated herein by reference): namely, CT-011 (pidilizumab; humanized IgG1; Curetech), MK-3475 (lambrolizumab, pembrolizumab; humanized IgG4; Merck), BMS-936558 (nivolumab; human IgG4; Bristol-Myers Squibb), AMP-224 (fusion protein of PD-L2 and IgG2a; AstraZeneca), BMS-936559 (human IgG4; Bristol-Myers Squibb), MEDI4736 (humanized IgG; AstraZeneca), MPDL3280A (human IgG; Genentech), MSB0010718C (human IgG1; Merck-Serono); or the antibody used in accordance with the present invention may be MEDI0680 (AMP-514; AstraZeneca), i.e., a humanized IgG4 mAb.

[0040] In certain embodiments, CT-011 antibody may be administered to humans at a dosage of 0.2 mg / kg to 6 mg / kg, or at a dosage between 1.5 mg / kg and 6 mg / kg; MK-3475 antibody may be administered to humans at a dosage of 1 mg / kg to 10 mg / kg; BMS-936558 may be administered to humans at a dosage of 0.3 mg / kg to 20 mg / kg, at a dosage of 0.3 mg / kg to 10 mg / kg, at a dosage of 1 mg / kg to 10 mg / kg, or at 1 mg / kg or 3 mg / kg; BMS-936559 may be administered to humans at a dosage of 0.3 mg / kg to 10 mg / kg; MPDL3280A may be administered to humans at a dosage of 1 mg / kg to 20 mg / kg; MEDI4736 may be administered to humans at a dosage of 0.1 mg / kg to 15 mg / kg; MSB0010718C may be administered to humans at a dosage of 1 mg / kg to 20 mg / kg.

[0041] The anti-CTLA-4 antibody may be tremelimumab (Pfizer), a fully human IgG2 monoclonal antibody, or ipilimumab, a fully human IgG1 monoclonal antibody.

[0042] The anti-killer cell immunoglobulin-like receptor (KIR) antibody may be lirilumab (BMS-986015; developed by Innate Pharma and licensed to Bristol-Myers Squibb), a fully human monoclonal antibody.

[0043] The anti-LAG-3 antibody is directed against lymphocyte activation gene-3. One such antibody that may be used in accordance with the present invention is the monoclonal antibody BMS-986016 (pembrolizumab; humanized IgG4; Merck).

[0044] The anti-4-1BB antibody may be PF-05082566 (Pfizer Oncology), i.e., a fully humanized IgG2 agonist monoclonal antibody, or urelumab (BMS-663513; Bristol-Myers Squibb), i.e., a fully human IgG4 monoclonal antibody (these target 4-1BB).

[0045] In certain embodiments, combinations of antibodies may be used, for example, but not limited to, the following may be used: CT-011 in combination with rituximab (trade names: Rituxan, MabThera and Zytux), a chimeric monoclonal antibody against protein CD20, for example, each at 3 mg / kg; BMS-936558 (e.g., 1 mg / kg) in combination with ipilimumab (e.g., at 3 mg / kg); or BMS-936558 (e.g., 1 mg / kg to 10 mg / kg) in combination with a multi-peptide vaccine restricted by HLA-A * 0201 (Weber et al., 2013). [Table 1]

[0046] In certain embodiments, the agent is a p300 inhibitor whose formula is listed in Table 1, i.e., C646 (4-(4-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid), C146 (4-hydroxy-3-(((2-(3-iodophenyl)benz[d]oxazol-5-yl)imino)methyl)benzoic acid) or C375 (2-chloro-4-(5-((2,4-dioxo-3-(2-oxo-2-(p-tolylamino)ethyl)thiazolidin-5-ylidene)methyl)furan-2-yl)benzoic acid). In particular, the p300 inhibitor is C646.

[0047] In certain embodiments, the small molecule inhibitor of the indoleamine-2,3-dioxygenase pathway may be Indoximod (NLG9189; NewLink Genetics), INCB024360 (Incyte), or NLG919 (NewLink Genetics).

[0048] The HIF-1 regulator may be M30, i.e., 5-[N-methyl-N-prop-2-ynylaminomethyl]-8-hydroxyquinoline as described by Zheng et al. (Zheng et al., 2015).

[0049] In certain embodiments, the agent can be derived from a broad range of antibiotics that target Gram-positive and Gram-negative bacteria and thereby promote the immunomodulation of Tregs. For example, it can be derived from vancomycin, which has been shown to target Gram-positive bacteria and reduce the levels / activity of Tregs (Brestoff & Artis, 2013; Smith et al., 2013).

[0050] In certain embodiments, the agent can be any copolymer that would cause downregulation of Tregs in a particular therapy (e.g., YFAK, VYAK, VWAK, VEAK, FEAK, FAK, VAK, or WAK, etc.). As used herein, the terms "Cop-1" and "copolymer 1" are used interchangeably.

[0051] The pharmaceutical composition of the present invention comprises a suitable amount of positively charged amino acids (e.g., lysine or arginine, etc.) in combination with negatively charged amino acids (e.g., glutamic acid or aspartic acid, etc.) in a (preferably smaller) amount, and, if necessary, in combination with uncharged neutral amino acids (e.g., alanine or glycine, etc.) that serve as fillers if necessary, and, if necessary, It may contain as an active agent a random copolymer that contains amino acids adapted to confer immunogenicity to the polymer (such as aromatic amino acids such as tyrosine or tryptophan) and that modulates the activity or level of Tregs. Such compositions may contain any of such copolymers disclosed in International Publication WO00 / 05250, the entire content of which is hereby incorporated herein by reference.

[0052] More specifically, the compositions for use in the present invention include at least one copolymer selected from the group consisting of random copolymers containing one amino acid selected from each of at least three of the following groups: (a) lysine and arginine; (b) glutamic acid and aspartic acid; (c) alanine and glycine; and (d) tyrosine and tryptophan.

[0053] The copolymers for use in the present invention can be composed of L-amino acids or D-amino acids or mixtures thereof. As is known to those skilled in the art, L-amino acids are present in most natural proteins. However, various D-amino acids are commercially available and can be used in place of some or all of the amino acids used to produce the terpolymers and other copolymers used in the present invention. In the present invention, the use of copolymers containing both D-amino acids and L-amino acids is contemplated, as well as copolymers consisting essentially of either L-amino acids or D-amino acids.

[0054] In certain embodiments, the pharmaceutical composition of the present invention consists essentially of the copolymer 1, i.e., a random polypeptide mixture of L-glutamic acid (E), L-alanine (A), L-tyrosine (Y), and L-lysine (K) in an approximate ratio of 1.5:4.8:1:3.6, having a net overall positive charge and a molecular weight of about 2 kDa to about 40 kDa. In certain embodiments, Cop1 has an average molecular weight of about 2 kDa to about 20 kDa, or about 4.7 kDa to about 13 kDa, or about 4 kDa to about 8.6 kDa, or about 5 kDa to about 9 kDa, or about 6.25 kDa to 8.4 kDa. In other embodiments, Cop1 has an average molecular weight of about 13 kDa to about 20 kDa, or about 13 kDa to about 16 kDa, or about 15 kDa to about 16 kDa. Other average molecular weights for Cop1 that are less than 40 kDa are also encompassed by the present invention. The copolymer 1 in the above molecular weight range can be prepared by methods known in the art, for example, by the process described in U.S. Patent No. 5,800,808, the entire contents of which are incorporated herein by reference in their entirety. Copolymer 1 may be a polypeptide containing about 15 to about 100 amino acids or about 40 to about 80 amino acids in length. In certain embodiments, Cop1 is in its acetate form, known by the common name glatiramer acetate, which is approved in several countries under the trade name Copaxone® (Teva Pharmaceuticals Ltd., Petach Tikva, Israel) for the treatment of multiple sclerosis (MS). The activity of copolymer 1 for the pharmaceutical compositions disclosed herein is expected to remain if one or more of the following substitutions are made: aspartic acid in place of glutamic acid, glycine in place of alanine, arginine in place of lysine, and tryptophan in place of tyrosine.

[0055] In certain embodiments of the present invention, the copolymer that regulates the activity or level of Tregs is a copolymer consisting of three different amino acids (where each is from a different one of three of groups (a) - (d)). These copolymers are referred to herein as terpolymers.

[0056] In one embodiment, the copolymer that regulates the activity or level of Tregs is a terpolymer containing tyrosine, alanine, and lysine (hereinafter referred to as YAK), provided that the average molar fraction of these amino acids can be varied: tyrosine can be present at a molar fraction of about 0.05 - 0.250, alanine can be present at a molar fraction of about 0.3 - 0.6, and lysine can be present at a molar fraction of about 0.1 - 0.5. The molar ratio of tyrosine, alanine, and lysine may be about 0.10:0.54:0.35, respectively. It is possible to substitute lysine with arginine, alanine with glycine, and / or tyrosine with tryptophan.

[0057] In certain embodiments, the copolymer that regulates the activity or level of Tregs is a terpolymer containing tyrosine, glutamic acid, and lysine (hereinafter referred to as YEK), provided that the average molar fraction of these amino acids can be varied: glutamic acid can be present at a molar fraction of about 0.005 - 0.300, tyrosine can be present at a molar fraction of about 0.005 - 0.250, and lysine can be present at a molar fraction of about 0.3 - 0.7. The molar ratio of glutamic acid, tyrosine, and lysine may be about 0.26:0.16:0.58, respectively. It is possible to substitute glutamic acid with aspartic acid, lysine with arginine, and / or tyrosine with tryptophan.

[0058] In certain embodiments, the copolymer that modulates the activity or level of Tregs is a terpolymer containing lysine, glutamic acid, and alanine (hereinafter referred to as KEA), provided that the average molar fraction of these amino acids can be varied: glutamic acid can be present at a molar fraction of about 0.005 to 0.300, alanine can be present at a molar fraction of about 0.005 to 0.600, and lysine can be present at a molar fraction of about 0.2 to 0.7. The molar ratio of glutamic acid, alanine, and lysine may be about 0.15:0.48:0.36, respectively. It is possible to substitute glutamic acid with aspartic acid, alanine with glycine, and / or lysine with arginine.

[0059] In certain embodiments, the copolymer that modulates the activity or level of Tregs is a terpolymer containing tyrosine, glutamic acid, and alanine (hereinafter referred to as YEA), provided that the average molar fraction of these amino acids can be varied: tyrosine can be present at a molar fraction of about 0.005 to 0.250, glutamic acid can be present at a molar fraction of about 0.005 to 0.300, and alanine can be present at a molar fraction of about 0.005 to 0.800. The molar ratio of glutamic acid, alanine, and tyrosine may be about 0.21:0.65:0.14, respectively. It is possible to substitute tyrosine with tryptophan, glutamic acid with aspartic acid, and / or alanine with glycine.

[0060] The average molecular weight of the above terpolymers (YAK, YEK, KEA, and YEA) can be varied between about 2 kDa and 40 kDa, preferably between about 3 kDa and 35 kDa, more preferably between about 5 kDa and 25 kDa.

[0061] The copolymers 1 and other copolymers that regulate the activity or level of Tregs may be prepared by methods known in the art, for example, by methods under condensation conditions using amino acids in a desired molar ratio in solution, or by solid-phase synthesis procedures. The condensation conditions include appropriate temperature, pH, and solvent conditions for condensing the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond. Various condensing agents (e.g., dicyclohexylcarbodiimide) can be used to facilitate the formation of peptide bonds. Various blocking groups can be used to protect functional groups (e.g., side chain moieties and some amino or carboxyl groups) from unwanted side reactions.

[0062] For example, the above copolymers can be prepared by the process disclosed in U.S. Patent No. 3,849,550, in which the N-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate, and Nε-trifluoroacetyl-lysine are polymerized in anhydrous dioxane at ambient temperature (20 °C to 26 °C) using diethylamine as an initiator. The γ-carboxyl group of glutamic acid can be deblocked with hydrogen bromide in glacial acetic acid. The trifluoroacetyl group is removed from lysine with 1 M piperidine. Those skilled in the art can readily understand that this process can be adjusted to produce peptides and polypeptides containing the desired amino acids, i.e., three of the four amino acids in copolymer 1, by selectively excluding the reactions associated with any one of glutamic acid, alanine, tyrosine, or lysine.

[0063] The molecular weight of the above copolymer can be adjusted during the polypeptide synthesis or after the copolymer has been produced. To adjust the molecular weight during the polypeptide synthesis, the synthesis conditions or the amount of amino acids are adjusted, and as a result, when the polypeptide reaches the desired approximate length, the synthesis is stopped. After the synthesis, the polypeptide having the desired molecular weight can be obtained by any available size selection procedure, such as chromatography of the polypeptide on a column or gel for molecular weight size fractionation, and recovery of the desired molecular weight range. The above copolymer can also be partially hydrolyzed, for example, by acid hydrolysis or enzymatic hydrolysis to remove high molecular weight species, and then purified to remove the acid or enzyme.

[0064] In one embodiment, a copolymer having the desired molecular weight may be prepared by a process that includes reacting a protected polypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptide having the desired molecular weight profile. The reaction is carried out at a time and temperature pre-determined by one or more test reactions. During the test reaction, the time and temperature are varied and the molecular weight range of a given batch of the test polypeptide is determined. The test conditions that give the optimal molecular weight range to that batch of the polypeptide are used for that batch. Thus, a trifluoroacetyl-polypeptide having the desired molecular weight profile can be produced by a process that includes reacting a protected polypeptide with hydrobromic acid at a time and temperature pre-determined by a test reaction. The trifluoroacetyl-polypeptide having the desired molecular weight profile is then further treated with an aqueous solution of piperidine to form a low-toxicity polypeptide having the desired molecular weight.

[0065] In certain embodiments, a test sample of a protected polypeptide derived from a given batch is reacted with hydrobromic acid at a temperature of about 20°C to 28°C for about 10 hours to 50 hours. The optimal conditions for that batch are determined by performing several test reactions. For example, in one embodiment, the protected polypeptide is reacted with hydrobromic acid at a temperature of about 26°C for about 17 hours.

[0066] Since the binding motifs of Cop1 to MS-related HLA-DR molecules are known (Fridkis-Hareli et al., 1999), polypeptides having defined sequences can be readily prepared and tested for binding to the peptide-binding groove of HLA-DR molecules as described in the publication by Fridkis-Hareli et al. (1999). Examples of such peptides are those disclosed in International Publications WO00 / 05249 and WO00 / 05250 (the entire contents of which are incorporated herein by reference), and examples of such peptides include the peptides of SEQ ID NOs: 1 to 32 (Table 2). Such peptides and other similar peptides would be expected to have activities similar to Cop1. Such peptides and other similar peptides are also considered to be within the definition of copolymers that cross-react with CNS myelin antigens, and their use is considered to be part of the present invention.

[0067]

Table 2

[0068] According to the present invention, the definition of a "copolymer that modulates the activity or level of Tregs" is meant to include other synthetic amino acid copolymers, such as the random 4 - amino acid copolymers described by Fridkis - Hareli et al. (2002) and U.S. Patent No. 8,017,125 (as candidates for treating multiple sclerosis), namely, the following copolymers: VFAK copolymers containing the amino acids valine (V), phenylalanine (F), alanine (A), and lysine (K); VYAK copolymers containing the amino acids valine (V), tyrosine (Y), alanine (A), and lysine (K); VWAK copolymers containing the amino acids valine (V), tryptophan (W), alanine (A), and lysine (K); VEAK copolymers containing the amino acids valine (V), glutamic acid (E), alanine (A), and lysine (K); FEAK copolymers containing the amino acids phenylalanine (F), glutamic acid (E), alanine (A), and lysine (K); FAK copolymers containing the amino acids phenylalanine (F), alanine (A), and lysine (K); VAK copolymers containing the amino acids valine (V), alanine (A), and lysine (K); and WAK copolymers containing the amino acids tryptophan (W), alanine (A), and lysine (K).

[0069] The pharmaceutical composition according to the present invention may be for treating a neurodegenerative disease, disorder or condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis; secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, a retinal degenerative disorder selected from the group consisting of age - related macular degeneration and retinitis pigmentosa; anterior ischemic optic neuropathy; glaucoma; uveitis; depression; trauma - related stress or post - traumatic stress disorder, frontotemporal dementia, Lewy body dementia, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia or progressive supranuclear palsy. In certain embodiments, the CNS condition is age - related dementia.

[0070] In certain embodiments, the CNS condition is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease.

[0071] The pharmaceutical composition according to the present invention may further be for treating CNS injury selected from spinal cord injury, closed head injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebral ischemia, optic nerve injury, myocardial infarction, organophosphate poisoning, and injury caused by tumor resection.

[0072] As described above, the inventors have found that the cognitive function in mice mimicking Alzheimer's disease is improved by the present invention. Thus, the pharmaceutical composition may be for use in improving the motor and / or cognitive function of the CNS, for example, in individuals without a diagnosed disease, as well as in alleviating the age-related loss of cognitive function that may occur in people suffering from neurodegenerative diseases. Moreover, the pharmaceutical composition may be for use in alleviating the loss of cognitive function resulting from acute stress or traumatic episodes. The cognitive function referred to herein may include learning, memory, or both.

[0073] As used herein, the term "CNS function" refers, inter alia, to receiving and processing sensory information, thinking, learning, remembering, recognizing, speaking and understanding language, controlling motor function as well as auditory and visual responses, maintaining balance and equilibrium, coordinating movement, transmitting sensory information, and controlling autonomic functions such as breathing, heart rate, and digestion.

[0074] The terms "cognition," "cognitive function," and "cognitive performance" are used interchangeably herein It is associated with any mental process or mental state that involves the use of, learning, memory, creation of mental images, thinking, recognition, reasoning, spatial ability, speech and language skills, language acquisition, and the ability for judgment and attention. Cognition is formed in many regions of the brain, such as in the hippocampus, cortex, and other brain structures. However, long-term memory is thought to be stored at least in part in the cortex, and it is known that sensory information is acquired, fixed, and retrieved by certain cortical structures (taste area) within the insular cortex.

[0075] In humans, cognitive function may be measured in any known way, for example, and without limitation, by the Clinician's Interview-Based Impression of Change Plus scale (CIBIC-plus scale); the Mini-Mental State Examination (MMSE); the Neuropsychiatric Inventory (NPI); the Clinical Dementia Rating scale (CDR); the Cambridge Neuropsychological Test Automated Battery (CANTAB) or the Sandoz Clinical Assessment-Geriatric (SCAG). Cognitive function may also be measured indirectly using various imaging techniques, for example, positron emission tomography (PET), functional magnetic resonance imaging (fMRI), single photon emission computed tomography (SPECT), or any other imaging technique that enables measurement of brain function.

[0076] Improvement of one or more processes that affect cognition in a patient would mean improvement of cognitive function in said patient, and thus, in certain embodiments, improving cognition includes improving learning, plasticity, and / or long-term memory. The terms "improve" and "enhance" may be used interchangeably.

[0077] The term "learning" relates to the acquisition or attainment of new knowledge, behaviors, skills, values or preferences, or to the modification and enhancement of existing knowledge, behaviors, skills, values or preferences.

[0078] The term "plasticity" relates to synaptic plasticity, brain plasticity or neural plasticity associated with the ability of the brain to change through learning and the ability to change already acquired memories. One measurable parameter that reflects plasticity is memory erasure.

[0079] The term "memory" relates to the process by which information is encoded, stored, and retrieved. Memory has three distinguishable categories: sensory memory, short-term memory, and long-term memory.

[0080] The term "long-term memory" refers to the ability to retain information over a long or indefinite period. Long-term memory includes two main parts: explicit memory (declarative memory) and implicit memory (non-declarative memory). Long-term memory is achieved by memory consolidation, a set of processes that stabilize memory traces after their initial acquisition. Consolidation is distinguished into two specific processes: synaptic consolidation (which occurs within the first few hours after learning) and systemic consolidation (where hippocampus-dependent memories become independent of the hippocampus over a period of weeks to years).

[0081] In a further aspect, the present invention is a method for treating a central nervous system (CNS) disease, disorder, condition or injury that does not include relapsing-remitting multiple sclerosis (RRMS), an autoimmune neuroinflammatory disease, the method comprising administering to an individual in need thereof a pharmaceutical composition according to the present invention as defined above, wherein the pharmaceutical composition is administered according to a dosing schedule that includes at least two treatment cycles (each treatment cycle in turn including a treatment period and then an intervening period).

[0082] In certain embodiments, the treatment period comprises administering the pharmaceutical composition to the individual, and the treatment period is maintained until at least the level is lower than a reference standard, and administration is discontinued during the period of the intermission, and the intermission is maintained as long as the level is lower than the reference standard. The reference standard may be selected from (a) the level of the systemic presence or activity of regulatory T cells or myeloid-derived suppressor cells measured in the most recent blood sample obtained from the individual prior to said administration, or (b) the level of the systemic presence or activity of regulatory T cells or myeloid-derived suppressor cells characteristic of a population of individuals suffering from a CNS disease, disorder, condition or injury.

[0083] Alternatively, the treatment period comprises administering the pharmaceutical composition to the individual, and the treatment period is maintained until at least the systemic presence or level of IFNγ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation exceeds a reference standard, and administration is discontinued during the period of the intermission, and the intermission is maintained as long as the level exceeds the reference standard, provided that in this case the reference standard is selected from (a) the level of the systemic presence or activity of IFNγ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation measured in the most recent blood sample obtained from the individual prior to said administration, or (b) the level of the systemic presence or activity of IFNγ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation characteristic of a population of individuals suffering from a CNS disease, disorder, condition or injury.

[0084] The above-described embodiments that describe various features of the pharmaceutical composition of the present invention are relevant for the method of the present invention as well, since the same pharmaceutical composition is used in the method of the present invention.

[0085] In a further aspect, the present invention provides a pharmaceutical composition for use in treating a CNS disease, disorder, condition or injury that does not include RRMS, an autoimmune neuroinflammatory disease, the pharmaceutical composition comprising an active agent that causes a reduction in the level of systemic immunosuppression in an individual, selected from the following: (i) antibodies specific to the following: (a) CD47, (b) OX40, (c) VEGF-A (bevacizumab), (d) CD25, (e) GITR (GITR-induced mAb (DTA-1)), (f) CCR4, or (g) TIM-3 / galectin-9, (h) anti-killer cell immunoglobulin-like receptor, (i) anti-LAG-3, or (j) anti-4-1BB; (ii) any combination of (a) to (j); (iii) any combination of (a) to (j) in combination with an adjuvant (e.g., a TLR9 ligand (e.g., CpG, etc.)); (iv) proteins selected from the following: (a) the glycoprotein of the leaves of indendane (NLGP) or (b) sCTLA-4; (v) small molecules selected from the following: (a) sunitinib, (b) polyoxometalate-1 (POM-1), (c) α,β-methylene adenosine 5'-diphosphate (APCP), (d) arsenic trioxide (As2O3), (e) GX15-070 (obatoclax), (f) a retinoic acid antagonist, e.g., Ro41-5253 or LE-135, etc., (g) a SIRPα (CD47) antagonist, e.g., CV1-hIgG4 (as a single agent or in combination with an anti-CD47 antibody), (h) a CCR4 antagonist, e.g., AF399 / 420 / 18025 (as a single agent or in combination with an anti-CCR4 antibody), or (i) an adenosine A2B receptor antagonist, e.g., PSB603, etc.; (j) an antagonist of indoleamine-2,3-dioxygenase, (k) an HIF-1 regulator; (vi) silencing molecules, e.g., miR-126 antisense and anti-galectin-1 (Gal-1), etc.; (vii) OK-432, (viii) a combination of IL-12 and anti-CTLA-4; (ix) an antibiotic, e.g., vancomycin, etc.; or (x) any combination of (i) to (ix).

[0086] The pharmaceutical composition for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to its recipient.

[0087] The following examples of carriers, modes of administration, dosage forms, etc. are listed as known possibilities where carriers, modes of administration, dosage forms, etc. may be selected for use with the present invention. However, those skilled in the art will understand that any formulation and mode of administration, the selected given formulation and mode of administration must first be tested to determine that it achieves the desired result.

[0088] Modes of administration include, but are not limited to, parenteral routes such as intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, intraoral, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic or local.

[0089] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active agent is administered together. Carriers in pharmaceutical compositions may include binders such as microcrystalline cellulose, polyvinylpyrrolidone (povidone or povidone), tragacanth gum, gelatin, starch, lactose or lactose monohydrate; disintegrants such as alginic acid and corn starch; lubricants or surfactants such as magnesium stearate or sodium lauryl sulfate; and flow promoters such as colloidal silicon dioxide.

[0090] For oral administration, the pharmaceutical preparation may be in liquid form (e.g., solution, syrup or suspension), or may be provided as a drug product for reconstitution before use with water or other suitable vehicle. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives (e.g., suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters or fractionated vegetable oils) and preservatives (e.g., methyl p-hydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid), etc.). The pharmaceutical composition may take the form of tablets or capsules prepared by conventional means using, for example, pharmaceutically acceptable excipients (e.g., binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate) or wetting agents (e.g., sodium lauryl sulfate), etc.). Tablets may be coated by methods well known in the art.

[0091] Various preparations for oral administration may be suitably formulated to provide controlled release of the active compound.

[0092] For buccal administration, the composition may take the form of tablets or troches formulated in a conventional manner.

[0093] The composition may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. The injectable formulation may be provided in unit dosage form in, for example, an ampoule or a multi-dose container with added preservatives. The composition may take the form of a suspension, solution or emulsion in an oily vehicle or an aqueous vehicle, and may also contain various formulating agents, such as suspending agents, stabilizers and / or dispersing agents and the like. Alternatively, the active ingredient may be in powder form for constitution before use with a suitable vehicle (for example, sterile pyrogen-free water).

[0094] The composition may also be formulated in a rectal composition (for example, suppositories or retention enemas) containing, for example, conventional suppository bases (such as cocoa butter or other glycerides).

[0095] For administration by inhalation, the composition for use according to the present invention is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer by the use of a suitable propellant (for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (for example, made of gelatin) for use in an inhaler or insufflator may be formulated containing the compound and a suitable powder base (for example, lactose or starch).

[0096] The determination of the dosage of the active ingredient for use in humans is based on the generally used practices in this technical field and will ultimately be determined by a physician in clinical trials. The approximately equivalent dosage expected for human administration can be calculated using known formulas based on the evidence of in vivo experiments disclosed below in this specification (e.g., Reagan-Show et al. (2007), Reconsidered dose conversion from animal studies to human studies, The FASEB Journal, 22:659-661). According to this theoretical framework, the adult equivalent dosage (mg / kg body weight) is equal to the dosage (mg / kg body weight) given to mice multiplied by 0.081.

[0097] The present invention is next illustrated by the following non-limiting examples.

Examples

[0098] Materials and Methods Animals. Transgenic 5XFAD mice (Tg6799) that overexpress the transgenes of the familial AD mutant forms of human APP (Swedish mutation, K670N / M671L; Florida mutation, I716V; and London mutation, V717I) and the familial AD mutant form of PS1 (M146L / L286V) under the transcriptional control of the neuron-specific mouse Thy-1 promoter (Oakley et al., 2006), and AD double transgenic B6.Cg-Tg(APPswe, PSEN1dE9)85Dbo / J mice (Borchelt et al., 1997) were purchased from The Jackson Laboratory. Genotyping was performed by PCR analysis of tail DNA as previously described (Oakley et al., 2006). Heterozygous mutant cx3cr1 GFP / + mice (Jung et al., 2000) (B6.129P-cx3cr1 tm1Litt / J, which is one of the CX3CR1 chemokine receptor alleles replaced by a gene encoding GFP) were used as donors for BM chimeras. Foxp3.LuciDTR mice (Suffner et al., 2010) were mated with 5XFAD mice to obtain Foxp3+ Conditional depletion of Tregs was enabled. Animals were bred and maintained by the Animal Breeding Center of the Weizmann Institute of Science. All experiments detailed herein were in accordance with the rules established by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute of Science.

[0099] RNA purification, cDNA synthesis, and quantitative real-time PCR analysis. Total RNA from the hippocampal dentate gyrus (DG) was extracted using TRI Reagent (Molecular Research Center) and lysates were processed using the RNeasy Kit (Qiagen). They were purified. Total RNA from the choroid plexus was extracted using an RNA MicroPrep Kit (Zymo Research). mRNA (1 μg) was converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The expression of specific mRNAs was assayed using fluorescence-based quantitative real-time PCR (RT-qPCR). The RT-qPCR reaction was performed using a Fast-SYBR PCR Master Mix (Applied Biosystems). The quantification reaction was performed in triplicate for each sample using the standard curve method. Peptidylprolyl isomerase A (ppia) was selected as a reference (housekeeping) gene. The amplification cycle was 95°C for 5 seconds, 60°C for 20 seconds, and 72°C for 15 seconds. When the assay was completed, a melting curve was generated to evaluate the specificity of the reaction. For gene analysis of ifn-γ and ppia, cDNA was pre-amplified with 14 PCR cycles using non-random PCR primers according to the manufacturer's protocol (PreAmp Master Mix Kit; Applied Biosystems), thereby increasing the sensitivity of subsequent real-time PCR analysis. mRNA expression was determined using TaqMan RT-qPCR according to the manufacturer's instructions (Applied Biosystems). All RT-qPCR reactions were performed and analyzed using StepOne software V2.2.2 (Applied Biosystems). The following TaqMan Assays-on-Demand (trademark) probes were used: Mm02342430_g1 (ppia) and Mm01168134_m1 (ifn-γ). For all other genes examined, the following primers were used: ppia forward 5'-AGCATACAGGTCCTGGCATCTTGT-3' (SEQ ID NO: 33) and reverse 5'-CAAAGACCACATGCTTGCCATCCA-3' (SEQ ID NO: 34); icam1 forward 5'-AGATCACATTCACGGTGCTGGCTA-3' (SEQ ID NO: 35) and reverse 5'-AGCTTTGGGATGGTAGCTGGAAGA-3' (SEQ ID NO: 36);Forward of vcam1: 5’-TGTGAAGGGATTAACGAGGCTGGA-3’ (SEQ ID NO: 37) and reverse: 5’-CCATGTTTCGGGCACATTTCCACA-3’ (SEQ ID NO: 38); Forward of cxcl10: 5’-AACTGCATCCATATCGATGAC-3’ (SEQ ID NO: 39) and reverse: 5’-GTGGCAATGATCTCAACAC-3’ (SEQ ID NO: 40); Forward of ccl2: 5’-CATCCACGTGTTGGCTCA-3’ (SEQ ID NO: 41) and reverse: 5’-GATCATCTTGCTGGTGAATGAGT-3’ (SEQ ID NO: 42); Forward of tnf-γ: 5’-GCCTCTTCTCATTCCTGCTT-3’ (SEQ ID NO: 43) and reverse: CTCCTCCACTTGGTGGTTTG-3’ (SEQ ID NO: 44); Forward of il-1β: 5’-CCAAAAGATGAAGGGCTGCTT-3’ (SEQ ID NO: 45) and reverse: 5’-TGCTGCTGCGAGATTTGAAG-3’ (SEQ ID NO: 46); Forward of il-12p40: 5’-GAAGTTCAACATCAAGAGCA-3’ (SEQ ID NO: 47) and reverse: 5’-CATAGTCCCTTTGGTCCAG-3’ (SEQ ID NO: 48); Forward of il-10: 5’-TGAATTCCCTGGGTGAGAAGCTGA-3’ (SEQ ID NO: 49) and reverse: 5’-TGGCCTTGTAGACACCTTGGTCTT-3’ (SEQ ID NO: 50); Forward of tgfβ2: 5’-AATTGCTGCCTTCGCCCTCTTTAC-3’ (SEQ ID NO: 51) and reverse: 5’-TGTACAGGCTGAGGACTTTGGTGT-3’ (SEQ ID NO: 52); Forward of igf-1: 5’-CCGGACCAGAGACCCTTTG (SEQ ID NO: 53) and reverse: 5’-CCTGTGGGCTTGTTGAAGTAAAA-3’ (SEQ ID NO: 54); Forward of bdnf: 5’-GATGCTCAGCAGTCAAGTGCCTTT-3’ (SEQ ID NO: 55) and reverse: 5’-GACATGTTTGCGGCATCCAGGTAA-3’ (SEQ ID NO: 56);

[0100] Immunohistochemistry. Tissue processing and immunohistochemistry were performed on paraffin-embedded sections of mouse brain (6 μm thick) and human brain (10 μm thick). For human ICAM-1 staining Primary mouse anti-ICAM antibody (1:20; Abcam; ab2213) was used. Slide glass specimens were incubated with 3% H2O2 for 10 minutes, and secondary biotin-conjugated anti-mouse antibody was used, followed by biotin / avidin amplification with the Vectastain ABC kit (Vector Laboratories). Subsequently, 3,3'-diaminobenzidine (DAB substrate) (Zytomed kit) was added; the slide glass specimens were dehydrated and mounted with xylene-based mounting medium. For tissue staining, mice were subjected to transcardial perfusion with PBS before tissue excision and fixation. CP tissue was isolated from the lateral, third, and fourth ventricles of the brain under a dissecting microscope (Stemi DV4; Zeiss). For whole-mounted CP staining, tissues were fixed with 2.5% paraformaldehyde (PFA) at 4°C for 1 hour and subsequently transferred to PBS containing 0.05% sodium azide. Prior to staining, dissected tissues were washed with PBS and blocked at room temperature for 1 hour (20% horse serum, 0.3% Triton X-100, and PBS). Whole-mounted staining with primary antibody (in PBS containing 2% horse serum and 0.3% Triton X-100) or secondary antibody was performed at room temperature for 1 hour. After each step, three washes in PBS were performed. Tissues were placed on slide glasses, fixed with Immu-mount (obtained from 9990402, Thermo Scientific), and sealed with coverslips. For staining of sectioned brains, two different tissue preparation protocols (paraffin-embedded sections or floating sections sectioned with a microtome) were applied as previously described (Baruch et al., 2013; Kunis et al., 2013).The following primary antibodies were used: mouse anti-Aβ (1:300, Covance, #SIG-39320); rabbit anti-GFP (1:100, MBL, #598); rat anti-CD68 (1:300, eBioscience, #14-0681); rat anti-ICAM-1 (1:200, Abcam, #AB2213); goat anti-GFP (1:100, Abcam, #ab6658); rabbit anti-IBA-1 (1:300, Wako, #019-19741); goat anti-IL-10 (1:20, R&D systems, #AF519); rat anti-Foxp3 (1:20, eBioscience, #13-5773-80); rabbit anti-CD3 (1:500, Dako, #IS503); mouse anti-ZO-1, mouse anti-E-cadherin and rabbit anti-claudin-1 (all 1:100, Invitrogen, #33-9100, #33-4000, #51-9000); rabbit anti-GFAP (1:200, Dako, #Z0334). The secondary antibodies included the following: Cy2 / Cy3 / Cy5-conjugated donkey anti-mouse / goat / rabbit / rat antibodies (1:200; all obtained from Jackson Immunoresearch). Slide glass specimens were exposed to Hoechst nuclear staining for 1 minute (1:4000; Invitrogen Probes). Two negative controls were routinely used in the immunostaining procedure, namely, staining with an isotype control antibody followed by a secondary antibody, or staining with the secondary antibody alone. For intracellular staining of Foxp3, antigen retrieval from paraffin-embedded slide glass specimens was performed using a Retreivagen Kit (#550524, #550527; BD Pharmingen (trademark)). Microscopic analysis was performed using a fluorescence microscope (E800; Nikon) or a laser scanning confocal microscope (Carl Zeiss, Inc.). The fluorescence microscope was equipped with a digital camera (DXM 1200F; Nikon) and either a 20x NA 0.50 objective lens or a 40x NA 0.75 objective lens (Plan Fluor; Nikon). The confocal microscope was equipped with LSM510 laser scanning capability (three laser lights: Ar 488, HeNe 543, and HeNe 633).Records were performed on post-fixed tissues using acquisition software (NIS-Elements, F3 [Nikon] or LSM [Carl Zeiss, Inc.]). For quantification of staining intensity, as previously described (Burgess et al., 2010), total staining of cells and background was measured using ImageJ software (NIH), and the intensity of specific staining was calculated. Images were processed using Photoshop CS6 13.0 (Adobe) for removal of unwanted parts, merging and optimization of images, and were arranged using Illustrator CS5 15. .1 (Adobe).

[0101] Paraffin-embedded sections of human CP. Human brain sections from postmortem CNS non-diseased individuals of young and old age, as well as human brain sections from AD patients, were obtained from the Oxford Brain Bank (previously known as the Thomas Willis Oxford Brain Collection (TWOBC)) with appropriate consent and ethical committee approval (TW220). Experiments involving these sections were approved by the Weizmann Institute of Science Institutional Review Board.

[0102] Flow cytometry, sample preparation and analysis. Mice were subjected to transcardial perfusion with PBS and tissues were processed as previously described (Baruch et al., 2013). The brain was dissected and various brain regions were removed into PBS under a dissecting microscope (Stemi DV4; Zeiss), and the tissues were dissociated using a gentleMACS™ dissociator (Miltenyi Biotec). Choroid plexus tissue was isolated from the lateral, third and fourth ventricles of the brain and incubated in PBS (Ca / Mg 2+ containing 400 U / ml of type IV collagenase (Worthington Biochemical 2+Incubated at 37°C for 45 minutes (with accompaniment), and then homogenized by hand pipetting. The spleen was ground with the plunger of a syringe and treated with ACK (ammonium chloride potassium) lysis buffer to remove red blood cells. In all cases, samples were stained according to the manufacturer's protocol. All samples were filtered through a 70 μm nylon mesh and blocked with anti-Fc CD16 / 32 (1:100; BD Biosciencies). For intracellular staining of IFN-γ, cells were incubated with para-methoxyamphetamine (10 ng / ml; Sigma-Aldrich) and ionomycin (250 ng / ml; Sigma-Aldrich) for 6 hours, and brefeldin-A (10 μg / ml; Sigma-Aldrich) was added for the last 4 hours. Intracellular labeling of cytokines was performed using the BD Cytofix / Cytoperm™ Plus fixation / permeabilization kit (cat. no. 555028). For Treg staining, the FoxP3 staining buffer set from eBiosciences (cat. no. 00-5523-00) was used. The following fluorescent dye-labeled monoclonal antibodies were purchased from BD Pharmingen, BioLegend, R&D Systems or eBiosciences and used according to the manufacturer's protocol: PE or Alexa Fluor450 conjugated anti-CD4; PE conjugated anti-CD25; PerCP-Cy5.5 conjugated anti-CD45; FITC conjugated anti-TCRβ; APC conjugated anti-IFN-γ; APC conjugated anti-FoxP3; Brilliant Violet conjugated anti-CD45. Cells were analyzed on an LSRII cytometer (BD Biosciences) using FlowJo software. In each experiment, relevant negative control groups, positive controls and single-stained samples for each tissue were used to identify the population of interest and exclude other populations.

[0103] Preparation of BM chimeras. BM chimeras were prepared as previously described (Shechter et al., 2009; Shechter et al., 2013). Briefly, gender-matched recipient mice were subjected to lethal total-body irradiation (950 rad) with a head shield (Shechter et al., 2009). Mice were then transfected with CX3CR1 GFP / + 5 x 10 from donor 6 BM cells were injected intravenously. Mice were left for 8-10 weeks after BM transplantation to allow for reconstitution of hematopoietic lineages prior to use in experiments. The percentage of chimerism was assessed by the expression of circulating monocytes (CD11b + GFP-expressing cells in the CNS were determined by FACS analysis of blood samples according to the percentage of GFP-expressing cells in the CNS. In this head-occlusion model, chimerism of 60% was achieved on average, and GFP expression in the CNS was significantly increased. + Myeloid cells express CD45 high / CD11b high This indicates that the monocyte-derived macrophages, not microglia, (Shechter et al., 2013).

[0104] Morris water maze. Mice were given three trials per day for four consecutive days to learn to find a hidden platform located 1.5 cm below the water surface in a pool (1.1 m in diameter). The water temperature was maintained between 21°C and 22°C. The water was made opaque with powdered milk. In the test chamber, only distal visual shape cues and object cues were available to the mice to help locate the submerged platform. Escape latency, i.e., the time required to find the platform and climb onto it, was recorded with a maximum of 60 seconds. Each mouse was allowed to stay on the platform for 15 seconds and then was removed from the maze and returned to its home cage. If the mouse did not find the platform within 60 seconds, the mouse was placed on the platform by hand and returned to its home cage after 15 seconds. The inter-trial interval for each mouse was 10 minutes. On the fifth day, the mice were given a single 60-second trial without available escape, except for the platform. On the sixth and seventh days, the platform was placed in the quadrant opposite the first training quadrant, and the mice were given three retraining sessions per day. Data were recorded using an Etho Vision V7.1 automated tracking system (Noldus Information Technology). Statistical analysis was performed using analysis of variance (ANOVA) and Bonferroni post hoc tests. All MWM tests were conducted between 10:00 am and 5:00 pm during the light-off period.

[0105] Radial arm water maze. Specifically, as previously described (Alamed et al., 2006), the radial arm water maze (RAWM) was used to test spatial learning and memory. Briefly, six stainless steel inserts were placed in the tank, forming six swimming arms that radiated out from an open central area. The escape platform was located at the end of one arm (the goal arm) 1.5 cm below the water surface in the pool (1.1 m in diameter). The water temperature was maintained between 21°C and 22°C. The water was made opaque with powdered milk. In the test room, only distal visual shape cues and object cues were available to the mice to help locate the submerged platform. The location of the goal arm remained constant for a given mouse. On day 1, mice were trained for 15 trials (at regular intervals over 3 hours): the trials were alternated between a visible platform and a hidden platform, and only the hidden platform was used in the last 4 trials. On day 2, mice were trained for 15 trials using the hidden platform. Entries into the wrong arm, or failure to select an arm within 15 seconds, were scored as errors. Spatial learning and memory were measured by calculating the number of arm entry errors or escape latency of the mice in each trial. Training data were analyzed as mean error or mean escape latency for a set of training consisting of three consecutive trials.

[0106] GA administration. Each mouse was subcutaneously (s.c.) injected with a total dose of 100 μg of GA (batch number P53640; Teva Pharmaceutical Industries, Petah Tiqva, Israel) dissolved in 200 μl of PBS. Mice were injected either according to weekly GA therapy (Butovsky et al., 2006) or daily GA administration (Figures 8 and 16). Mice were euthanized either 1 week after the last GA injection or 1 month after treatment, as indicated for each experiment.

[0107] Conditional ablation of Tregs. Diphtheria toxin (DTx; 8 ng / g body weight; Sigma) was intraperitoneally (i.p.) injected daily for 4 consecutive days into Foxp3.LuciDTR mice (Suffner et al., 2010). The efficiency of DTx was confirmed by flow cytometry analysis of immune cells in blood and spleen, whereby FoxP3-expressing GFP + CD 4 + Almost complete (more than 99%) depletion of Treg cells was achieved (Figure 4).

[0108] P300 inhibition. Inhibition of p300 in mice was performed as previously described (Liu et al., 2013). p300i (C646; Tocris Bioscience) was dissolved in DMSO and injected i.p. daily for 1 week (8.9 mg kg -1 d -1 , i.p.). DMSO was injected similarly into vehicle-treated mice.

[0109] ATRA treatment. Administration of all-trans retinoic acid (ATRA) to mice was performed as previously described (Walsh et al., 2014). ATRA (Sigma) was dissolved in DMSO and injected i.p. every other day for 1 week (8 mg kg -1 d -1 ). DMSO was injected similarly into vehicle-treated mice.

[0110] Isolation and quantification of soluble Aβ (sAβ) protein. Tissue homogenization and extraction of sAβ protein were performed as previously described (Schmidt et al., 2005). Briefly, the parenchymal tissue of the brain was dissected, snap-frozen, and stored at -80 °C until homogenization. Proteins were sequentially extracted from the samples to obtain separate fractions containing proteins with different solubilities. The samples were homogenized using a mortar and pestle in a Dounce homogenizer in 10 volumes of ice-cold tissue homogenization buffer containing 250 mM sucrose, 20 mM Tris base, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM ethylene glycol tetraacetic acid (pH 7.4). After 6 strokes, the homogenate was mixed 1:1 with 0.4% diethylamine (DEA) in 100 mM NaCl solution before an additional 6 strokes and then centrifuged at 135,000 g for 45 minutes at 4 °C. The supernatant (DEA-soluble fraction containing extracellular and cytosolic proteins) was collected and neutralized with 10% of 0.5 M Tris-HCl (pH 6.8). Aβ 1_40 and Aβ 1_42 were individually measured by enzyme-linked immunosorbent assay (ELISA) from the soluble fraction using commercially available kits (Biolegend; #SIG-38954 and #SIG-38956) according to the manufacturer's instructions.

[0111] Quantification of Aβ plaques. From each brain, 6-μm coronal sections were collected and eight sections per mouse (from four different predetermined depths across the entire target region (dentate gyrus or cerebral cortex)) were immunostained. Histogram-based segmentation of positive-staining pixels was performed using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA). The segmentation algorithm was manually applied to each image in the dentate gyrus region or cortical layer V, and the percentage of the area occupied by total Aβ immunostaining was determined. The number of plaques was quantified from the same 6-μm coronal brain sections. The number of plaques was shown as the average number of plaques per brain region. Prior to quantification, the sections were coded to conceal the identity of the experimental groups, and the plaque burden was quantified by an observer who was unaware of the identity of the experimental groups.

[0112] Statistical analysis. The specific tests used to analyze each experimental group are shown in the figure legends. Data were analyzed using two-sided Student's t-tests to compare between two groups, one-way ANOVA to compare several groups, and then the Newman–Keuls post hoc procedure for pairwise comparisons of groups after the null hypothesis was rejected (P < 0.05). Data from behavioral tests were analyzed using two-way repeated-measures ANOVA, and the Bonferroni post hoc procedure was used for pairwise comparisons in follow-up investigations. The sample size was selected based on sufficient statistical power based on the literature and past experience, and mice were assigned to experimental groups according to age, sex, and genotype. The researchers were blinded to the identity of the experimental groups during the experimental period and the outcome assessment period. All inclusion and exclusion criteria were established in advance according to IACUC guidelines. Results are shown as mean ± s.e.m. In the graphs, error bars on the y-axis represent s.e.m. Statistical calculations were performed using GraphPad Prism software (Graphpad Software, San Diego, CA).

[0113] Introduction. Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by a progressive loss of cognitive function and neuronal damage, amyloid-beta (Aβ) plaque formation, and chronic inflammation within the central nervous system (CNS) (Akiyama et al., 2000; Hardy & Selkoe, 2002). Under these conditions, circulating myeloid cells and CNS-resident myeloid cells, namely microglia, play non-redundant roles in alleviating the neuroinflammatory response (Britschgi & Wyss-Coray, 2007; Cameron & Landreth, 2010; Lai & McLaurin, 2012). Specifically, microglia are ultimately unable to eliminate Aβ deposits, while monocyte-derived macrophages (mo-MΦ) infiltrating the CNS play a beneficial role in restricting Aβ plaque formation and combating AD-like pathology (Butovsky et al., 2007; Koronyo-Hamaoui et al., 2009; Mildner et al., 2011; Simard et al., 2006; Town et al., 2008). The brain choroid plexus (CP) has been identified as a selective entry point for leukocyte entry into the CNS, as its epithelial layer forms the blood-CSF barrier (BCSFB) and enables the recruitment of mo-MΦ cells and T cells after nerve tissue damage (Kunis et al., 2013; Shechter et al., 2013). Here, the inventors hypothesized that in AD, the suboptimal recruitment of inflammatory-resolving immune cells to the affected parenchyma is the result of systemic immunodeficiency associated with CP entry dysfunction.

[0114] Example 1. Choroid plexus (CP) entry activity along disease progression in a mouse model of AD. We first examined CP activity along disease progression in the 5XFAD transgenic mouse model of AD (AD-Tg); these mice co-express five mutations associated with familial AD and develop cerebral Aβ pathology and gliosis at as early an age as 2 months (Oakley et al., 2006). We found that along the progression of disease pathology, CP in AD-Tg mice expressed significantly reduced levels of determinants of leukocyte homing and leukocyte trafficking, including icam1, vcam1, cxcl10, and ccl2 (which have been shown to be upregulated by CP in response to acute CNS injury and are required for trans-epithelial migration of leukocytes; Kunis et al., 2013; Shechter et al., 2013), compared to age-matched wild-type (WT) controls (Figure 1A). Immunohistochemical staining for the integrin ligand ICAM-1 confirmed its reduced expression by the CP epithelium in AD-Tg mice (Figure 1b). In addition, staining for ICAM-1 in human postmortem brains showed an age-related decline in its expression in the CP epithelium, consistent with our previous observations (Baruch et al., 2014), and in a quantitative assessment of this effect, a further decline was revealed in AD patients compared to elderly individuals without CNS disease (Figure 2A). Since induction of leukocyte trafficking determinants by CP is dependent on signaling of interferon (IFN)-γ in the epithelium (Kunis et al., 2013), we next examined whether the observed effects might reflect a loss of IFN-γ availability in the CP. Examination of the CP of 5XFAD AD-Tg mice by flow cytometric intracellular staining revealed a significantly reduced number of IFN-γ-producing cells in this compartment (Figure 2B), and quantitative real-time PCR (RT-qPCR) analysis confirmed lower mRNA expression levels of ifn-γ in the CP of AD-Tg mice compared to age-matched WT controls (Figure 2C).

[0115] Example 2. Functional relationship between Treg-mediated systemic immunosuppression, CP entry activity, and AD pathology. Regulatory T cells (Tregs) play a critically important role in suppressing the systemic effector immune response (Sakaguchi et al., 2008). The inventors hypothesized that Treg-mediated systemic immunosuppression affects IFN-γ availability in CP and thus focused on the involvement of Tregs in AD pathology. Consistent with previous reports of elevated Treg levels and suppressive activity in AD patients (Rosenkranz et al., 2007; Saresella et al., 2010; Torres et al., 2013), the increased levels of Foxp3 Treg frequency in splenocytes of 5XFAD AD-Tg mice were revealed along with disease progression when compared to their age-matched WT littermates (Figures 3A, 3B). To investigate the functional relationship between Treg-mediated systemic immunosuppression, CP entry activity, and AD pathology, the inventors crossed 5XFAD AD-Tg mice with Foxp3-diphtheria toxin receptor (DTR + ) mice, thereby enabling transient conditional in vivo depletion of Fox3 + Tregs in AD-Tg / DTR + mice by administration of diphtheria toxin (DTx) (Figure 4A). Transient depletion of Tregs resulted in increased mRNA expression of leukocyte trafficking molecules by the CP of AD-Tg / DTR + mice compared to DTx-treated AD-Tg / DTR - littermates (Figure 5A). Analysis of the long-term effects (3 weeks later) of transient Treg depletion on brain parenchymal tissue revealed accumulation of immune cells in the brain, including increased numbers of CD45 + / CD11b high myeloid cells (which represent infiltrating mo-MΦ; Shechter et al., 2013) and CD4 high T cells (Figure 5B). In addition, short-term and transient depletion of Tregs led to increased Foxp3 + among CD4 + T cells accumulating in the brain as evaluated by flow cytometry +Significantly enhanced Tregs were generated (Figure 5C, Figure 5D). RT-qPCR analysis of the hippocampus showed increased expression of foxp3 and il10 mRNAs (Figure 5E).

[0116] Since the accumulation of immunomodulatory cells at the site of brain pathology continues after short-term depletion of Tregs, the inventors next examined whether short-term depletion of Tregs causes long-term effects on brain function. The inventors observed a decrease in gliosis in the hippocampus (Figure 5F) and also observed decreased mRNA expression levels of pro-inflammatory cytokines (such as il-12p40 and tnf-α, etc.) (Figure 5G). Moreover, the brain Aβ plaque burden in the hippocampal dentate gyrus and cerebral cortex (layer 5), that is, in the two brain regions that show robust Aβ plaque pathology in 5XFAD AD-Tg mice (Oakley et al., 2006) decreased (Figure 6A, Figure 6B). By evaluating the effect on cognitive function using the Morris water maze (MWM) test, significant improvement in spatial learning and memory in AD-Tg / DTR + mice after Treg depletion was revealed compared to DTx-treated AD-Tg / DTR - age-matched mice, reaching a performance similar to that of WT mice (Figure 6C - Figure 6E). In summary, these data revealed that temporarily interrupting Treg-mediated systemic immunosuppression in AD-Tg mice results in the accumulation of inflammatory resolution cells in the brain, including mo-MΦ and Tregs, followed by the dissipation of the neuroinflammatory response, the elimination of Aβ, and the recovery of cognitive decline.

[0117] Example 3. Weekly administration of copolymer-1 reduces Treg-mediated systemic immunosuppression, improves CP entry activity, and alleviates AD pathology. To further demonstrate the causality of the inverse correlation between systemic immunosuppression, CP function, and AD pathology, the inventors next used the immunomodulatory compound glatiramer acetate (GA; also known as copolymer-1 or Copaxone®). Glatiramer acetate has been found to have a therapeutic effect in the APP / PS1 mouse model of AD in weekly administration therapy (Butovsky et al., 2006); this effect was functionally associated with the mobilization of mo-MΦ to the brain sites of the disease pathology (Butovsky et al., 2007). Here, the inventors first examined whether CP in APP / PS1 AD-Tg mice was also deficient with respect to IFN-γ expression levels, similar to their observations in 5XFAD AD-Tg mice. The inventors found that in APP / PS1 AD-Tg mice, IFN-γ levels in CP were decreased relative to age-matched WT controls (Figure 7A). These results prompted the inventors to examine whether the weekly GA treatment effect in APP / PS1 mice (Butovsky et al., 2006) could be reproduced in 5XFAD AD-Tg mice, and if so, whether the therapeutic effect would affect systemic Tregs and the activation of CP for mo-MΦ transport. Therefore, the inventors treated 5XFAD AD-Tg mice with weekly GA administration therapy over a 4-week period (schematically shown in Figure 8A). The inventors found that 5XFAD AD-Tg mice treated with weekly GA showed reduced neuroinflammation (Figures 8B - 8D) and improved cognitive performance, with the improvement persisting until 2 months after treatment (Figures 8E - 8I). When examining the effect of weekly GA on systemic immunity and CP by flow cytometry, the inventors found Foxp3 in splenocytes in APP / PS1 AD-Tg mice, IFN-γ levels in CP were decreased relative to age-matched WT controls (Figure 7A). These results prompted the inventors to examine whether the weekly GA treatment effect in APP / PS1 mice (Butovsky et al., 2006) could be reproduced in 5XFAD AD-Tg mice, and if so, whether the therapeutic effect would affect systemic Tregs and the activation of CP for mo-MΦ transport. Therefore, the inventors treated 5XFAD AD-Tg mice with weekly GA administration therapy over a 4-week period (schematically shown in Figure 8A). The inventors found that 5XFAD AD-Tg mice treated with weekly GA showed reduced neuroinflammation (Figures 8B - 8D) and improved cognitive performance, with the improvement persisting until 2 months after treatment (Figures 8E - 8I). When examining the effect of weekly GA on systemic immunity and CP by flow cytometry, the inventors found Foxp3 in splenocytes +We found that the Treg level was decreased (Figure 9A), and also found that IFN-γ-producing cells increased in the CP of treated 5XFAD AD-Tg mice and reached levels similar to those observed in WT type controls (Figure 9B). The increased levels of IFN-γ-expressing cells in the CP of mice treated weekly with GA were accompanied by upregulated epithelial expression of leukocyte transport molecules (Figure 9C).

[0118] To detect the entry of infiltrating mo-MΦ into the CNS, we used 5XFAD AD-Tg / CX3CR1 GFP / + bone marrow (BM) chimeric mice prepared using (head protection). In these chimeric mice, visualization of circulating (green fluorescent protein (GFP) + labeled) myeloid cells was possible (Shechter et al., 2009; Shechter et al., 2013). We found increased homing of GFP GFP / + mo-MΦ to the CP and adjacent ventricular space after weekly GA treatment when compared to vehicle-treated AD-Tg / CX3CR1 + controls (Figures 9D - 9E). Immunohistochemistry of brain parenchymal tissue revealed the presence of GFP + mo-MΦ accumulation at the sites of brain plaque formation (Figure 9F), and quantification of infiltrating myeloid cells showed an increased number of CD11b high CD45 high expressing cells by flow cytometry analysis of the hippocampus in AD-Tg non-chimeric mice (Figures 9G, 9H). Collectively, these results demonstrated a functional link between mo-MΦ recruitment to the sites of AD pathology, decreased systemic Treg levels, and IFN-γ-dependent activation of the CP.

[0119] Example 4. By directly interfering with Treg activity over a short period, CP entry activity is improved and AD pathology is alleviated. 4.1 Interference with Treg activity using small molecule histone acetyltransferase inhibitors. The above findings suggested that Treg-mediated systemic immunosuppression hinders the ability to combat AD pathology, while evoking functions thought to be attributable to Tregs in cancer immunotherapy. This is because in cancer immunotherapy, these cells prevent the immune system from initiating an effective anti-tumor response (Bos & Rudensky, 2012; Nishikawa & Sakaguchi, 2010). Therefore, the inventors considered that a treatment that directly interferes with Foxp3 + Treg cell activity might be beneficial in AD. The inventors examined p300i (C646 (Bowers et al., 2010)), a non-peptidic inhibitor of p300, a histone acetyltransferase that regulates Treg function (Liu et al., 2013): this inhibitor has been shown to affect Treg suppressive activity while leaving the protective T effector cell response intact (Liu et al., 2013). The inventors found that mice treated with p300i showed elevated levels of systemic IFN-γ-expressing cells in the spleen (Figure 10A) compared to vehicle (DMSO)-treated controls, and similarly also showed elevated levels of systemic IFN-γ-expressing cells in the CP (Figure 10B). The inventors next treated AD-Tg mice for 1 week with either p300i or vehicle and examined cerebral Aβ pla The Aβ plaque load in the brain was examined 3 weeks after the p300i treatment. By immunohistochemical analysis, a significant decrease in the Aβ plaque load in the brain was revealed in AD-Tg mice treated with p300i (Figs. 10C - 10E). The inventors also examined whether the effect on plaque pathology after a single treatment course would persist beyond 3 weeks and, if so, whether additional treatment courses would contribute to a long-lasting effect. Therefore, the inventors compared AD-Tg mice that received only a single p300i treatment course and were examined 2 months later with an age-matched group that received two treatment courses with a 1-month interval during this period (schematically shown in Fig. 10F). The inventors found that the decrease in the brain plaque load was evident even 2 months after a single treatment course, but was stronger in mice that received two treatment courses with a 1-month interval (Fig. 10G). Since impaired synaptic plasticity and memory in AD are associated with elevated brain levels of soluble Aβ 1_40 / Aβ 1_42 (sAβ) (Shankar et al., 2008), the inventors also measured sAβ levels after a single cycle or repeated cycles of p300i treatment. Again, the inventors found that both a single course and two courses (with a 1-month interval) were effective in reducing brain sAβ, and nevertheless, this effect was stronger after repeated courses with respect to the impact on sAβ 1_42 (Fig. 10H). These results indicate that a single short-term treatment course is effective, but repeated treatment courses would be advantageous for maintaining a long-lasting therapeutic effect similar to the inventors' observations after weekly GA treatment.

[0120] 4.2 Inhibition of Treg activity using anti-PD1 antibody. At 10 months of age, 5XFAD Alzheimer's disease (AD) transgenic (Tg) mice were injected i.p. with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a; #BE0089; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 of the experiment, and the mice were examined for their cognitive performance 3 weeks later by a spatial learning and memory task in the radial arm water maze (RAWM) as previously described in detail (Alamed et al., 2006) (schematically shown in Fig. 11A). Briefly, on day 1 of the RAWM task, the mice were trained for 15 trials (at regular intervals over 3 hours): the trials were alternated between a visible platform and a hidden platform, and only the hidden platform was used in the last 4 trials. On day 2, the mice were trained for 15 trials using the hidden platform. Entries into incorrect arms, or failure to select an arm within 15 seconds, were scored as errors. Spatial learning and memory were measured by calculating the number of arm entry errors or escape latency of the mice in each trial. Age-matched untreated WT-type mice and AD-Tg mice were used as controls. The inventors found that 5XFAD AD-Tg mice treated with a single treatment period including two injections of anti-PD1 (on days 1 and 4) showed significantly improved spatial cognitive performance in the RAWM when evaluated 3 weeks later (Fig. 11B).

[0121] Next, the inventors examined whether the impact on disease pathology was accompanied by a decrease in systemic immunosuppression. The inventors repeated the above experiment, but this time, the mice were examined when the treatment period ended (day 7 of the experiment) (schematically shown in Figure 12A). The inventors found that at this time point, the attenuation of systemic immunosuppression in AD-Tg mice treated with PD-1 was accompanied by a systemic effect of increased IFN-γ-producing CD4 splenocytes (Figure 12B), and that this effect correlated with a local effect in the CP of increased mRNA levels of IFN-γ (Figure 13A), and also correlated with an increase in the expression of the leukocyte transport molecules in the CP, the chemokines CCL2 and CXCL10 (Figure 13B). These data indicate that during the short-term anti-PD-1 treatment period in AD-Tg mice, as expected, there was a systemic response of weakening Treg-mediated immunosuppression (Naidoo et al., 2014), and activation of the CP entry activity for leukocyte transport to the CNS.

[0122] Finally, the inventors examined the impact on disease pathology in AD-Tg mice and investigated whether a further treatment period was beneficial in its impact on pathology. To achieve this goal, 10-month-old AD-Tg mice received either a single anti-PD-1 treatment period as described above or a further treatment with a 3-week interval. The control group was subjected to either treatment with IgG or no treatment, and all mouse groups were tested for their cognitive performance 3 weeks later (schematically shown in Figure 14A). The inventors found that AD-Tg mice treated with a single anti-PD-1 (「AD-Tg+PD-1 X1」), when examined 2 months later, showed significant cognitive improvement compared to IgG-treated and untreated AD-Tg mice, but the effect was less robust than when the same mice were evaluated for cognitive performance 1 month earlier. In contrast, AD-Tg mice that received another anti-PD-1 treatment period (「AD-Tg+PD-1 "X2") showed significantly better spatial learning and memory ability in the RAWM compared to AD-TG that received a single treatment period, similar to when compared to IgG-treated or untreated AD-Tg mice (Figure 14B). These findings revealed that repeated treatment periods are necessary to maintain a long-lasting therapeutic effect.

[0123] 4.3 Interference with Treg activity using a combination of anti-PD1 antibody and anti-CTLA4 antibody. At 10 months of age, 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg of anti-CTLA4 antibody (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089, or polyclonal golden hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.) were injected i.p. into 5XFAD Alzheimer's disease (AD) transgenic (Tg) mice on the first and fourth days of the experiment, and the mice were examined for their cognitive performance 3 weeks later by a spatial learning and memory task in the radial arm water maze (RAWM) as described above.

[0124] Some mice receive an additional treatment period with a 3-week intermission. The control group is subjected to either IgG treatment or no treatment, and all mouse groups are tested for their cognitive performance 3 weeks later.

[0125] Mice treated with the antibody combination are expected to show significant cognitive improvement compared to IgG-treated and untreated AD-Tg mice, and also to show a significant reduction in brain plaque burden.

[0126] Example 5. Enhancement of Treg activity has an adverse effect on AD pathology. To demonstrate the negative role of Treg-mediated systemic immunosuppression in AD, the inventors next examined whether enhancing systemic Treg levels might have the opposite adverse effect on AD pathology. To investigate this, the inventors enhanced Treg suppressive function in AD-Tg mice by administration of all-trans retinoic acid (ATRA): in this case, all-trans retinoic acid induces Treg differentiation (Mucida et al., 2007), stabilizes the Treg phenotype (Zhou et al., 2010), and renders Tregs more suppressive (Zhou et al., 2010). The inventors used 5XFAD AD-Tg mice at a relatively early stage of disease progression and treated them with either ATRA or vehicle (DMSO). AD-Tg mice treated with ATRA showed a significantly greater splenocyte frequency of Foxp3 + CD 25 + Tregs (Figures 15A, 15B). When the mice were examined 3 weeks after the last ATRA injection, greater cerebral Aβ plaque burden and gliosis were revealed (an approximately 2-fold to 3-fold increase; Figures 15C–15E), and evaluation of sAβ revealed increased sAβ 1_40 levels and sAβ 1_42 levels in the brain after enhancement of systemic Tregs (Figures 15F–15G). Evaluation of cognitive performance using RAWM showed a worsening of spatial memory deficit in ATRA-treated AD-Tg mice compared to vehicle-treated AD-Tg mice (Figure 15H).

[0127] Daily administration of GA is known to induce Tregs and, in light of our current findings of the negative impact of systemic Tregs on AD pathology, in conjunction with the fact that it is used clinically to treat multiple sclerosis (MS) (Haar et al., 2009; Hong et al., 2005; Weber et al., 2007), we examined whether daily GA in daily therapy (over a one-month period), as opposed to weekly GA, might have a negative impact on disease pathology in AD-Tg mice. We compared the effect of daily GA administration to weekly GA administration (schematically shown in FIG. 16A) in 5XFAD AD-Tg mice. Evaluation of cognitive performance by the RAWM task revealed that, in contrast to the beneficial effect of weekly GA treatment, neither a beneficial effect on spatial memory nor a tendency towards a worsening effect was observed in AD-Tg mice receiving daily GA (FIG. 16B). Additionally, unlike the robust effect of weekly GA administration on plaque clearance, AD-Tg mice treated with daily GA showed no beneficial effect or a moderate negative effect on plaque burden (FIGS. 16C-16F). These findings highlight how two CNS pathologies with neuroinflammation, MS and AD, can be affected in opposite and discriminative ways by the same immunomodulatory treatment with daily GA (Schwartz & Baruch, 2014a).

[0128] Example 6. By directly interfering with Treg activity, CP entry activity is improved and PTSD pathology is prevented or alleviated. Severe stress phenotypes or chronic stress can potentially cause post-traumatic stress disorder (PTSD) and depression. The inventors have previously suggested that CP entry activity may be very important for coping with mental stress, and that if the function of CP is not optimal, a traumatic mental episode may cause PTSD (Schwartz & Baruch, 2012). The inventors further hypothesized that timely systemic intervention after trauma would help modify the CP response while potentially preventing the development of the chronic state of PTSD. The inventors' finding that weakening Treg-mediated systemic immunosuppression for a short period has long-term effects on brain pathology suggests that this intervention would prevent the development of PTSD if administered immediately after the traumatic event.

[0129] To test the inventors' working hypothesis that CP is involved in coping with traumatic stress and that if traumatic stress causes the development of PTSD, CP may be dysfunctional, the inventors employed a physiological PTSD-like animal model: in this model, mice exhibit hypervigilant behavior, reduced attention, increased risk assessment, and sleep disturbances (Lebow et al., 2012). In this experimental model of PTSD induction, mice are acclimated to a reversed light-dark cycle for 10 days and given two episodes of electric shock (trauma and cue), which is referred to as "PTSD induction," and are evaluated at various time points after trauma. After the traumatic event, mice are injected with the compound that temporarily reduces peripheral immunosuppression. Mice are treated according to one or more of the following therapies: · Mice receive i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089; Bioxcell Lifesciences Pv t. LTD.) on days 1 and 4 after the traumatic event and are examined after a further 2-week intermission; · Mice were subjected to i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg of anti-CTLA4 antibody (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089, or polyclonal golden hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 of the experiment and examined after a 2-week intermission period; · Mice were subjected to i.p. injection with GA weekly as described above after the traumatic event and examined after a 2-week intermission period; · Mice were subjected to treatment with p300i or vehicle over the course of 1 week after the traumatic event and examined 3 weeks later as described above; Some mice receive additional treatment periods with appropriate intermission periods.

[0130] Mice receiving treatment are not expected to show anxiety behaviors associated with PTSD in this experimental model, as evaluated by the time spent in exploration and risk assessment in the light / dark maze or other behavioral tasks as described by (Lebow et al., 2012).

[0131] Example 7. Transient reduction of systemic immunosuppression alleviates the pathology of Parkinson's disease. Parkinson's disease (PD) transgenic (Tg) mice are used in these experiments. Mice are treated at the disease progression stage according to one or more of the following therapies: · Mice were subjected to i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 after the traumatic event and examined after a further 2-week intermission period; · Mice were subjected to i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg of anti-CTLA4 antibody (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089, or polyclonal golden hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 of the experiment and were examined after a 2-week interval; · Mice were subjected to i.p. injection with GA weekly as described above after the traumatic event and were examined after a 2-week interval; · Mice were subjected to treatment with p300i or vehicle over the course of 1 week after the traumatic event and were examined 3 weeks later as described above; Some mice receive an additional treatment period with an appropriate interval (about 3 weeks to 1 month).

[0132] The neurological function of movement is evaluated using, for example, the rotarod performance test, by which the ability of a mouse to stay on a rotating rod is evaluated.

[0133] PD-Tg mice treated with a single treatment period are expected to show significantly improved motor performance compared to the control group or untreated group treated with IgG or vehicle. PD-Tg mice that undergo two treatment courses and are examined after an appropriate interval are expected to show a long-lasting therapeutic effect. To maintain this therapeutic effect, mice are subjected to an effective treatment period with an appropriate interval during each treatment period. are subjected to an effective treatment period with an appropriate interval during each treatment period.

[0134] Example 8. Transient reduction of systemic immunosuppression alleviates the pathology of Huntington's disease. The models used in these experiments may be the Huntington's disease (HD) R6 / 2 gene recombinant mouse (Tg) test system. R6 / 2 gene recombinant mice overexpress a mutant human huntingtin gene containing a large number of CAG repeats in mice at the disease progression stage. These mice show progressive behavioral-motor disorders that begin as early as 5 to 6 weeks of age and cause premature death at 10 to 13 weeks. Symptoms include low body weight, clasping, tremors, and seizures.

[0135] Mice are treated at 45 days of age according to one or more of the following therapies: · Mice receive an i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 after the trauma event and are examined after a further 2-week interval; · Mice receive an i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg of anti-CTLA4 antibody (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089, or polyclonal golden hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 of the experiment and are examined after a further 2-week interval. · Mice receive an i.p. injection with GA weekly as described above after the trauma event and are examined after a 2-week interval; · Mice receive treatment with p300i or vehicle over a 1-week period after the trauma event and are examined 3 weeks later as described above; Some mice receive a further treatment period with an appropriate interval (about 3 weeks to 1 month).

[0136] The neurological function of movement is evaluated using, for example, the rotarod performance test, which evaluates the ability of a mouse to stay on a rotating rod.

[0137] HD-Tg mice treated with a single treatment period are expected to show significantly improved motor performance compared to control groups or untreated groups treated with IgG or vehicle. HD-Tg mice that undergo two treatment courses and are examined after an appropriate intermission period are expected to show a long-lasting therapeutic effect. To maintain this therapeutic effect, the mice are subjected to an effective treatment period with appropriate intermissions between each treatment period.

[0138] Example 9. Transient reduction of systemic immunosuppression alleviates the pathology of amyotrophic lateral sclerosis. The model used in this experiment may be a transgenic mouse (B6SJL-TgN(SOD1-G93A)1Gur, herein referred to as "ALS mouse") that overexpresses a mutant human SOD1 allele containing the Gly93→Ala (G93A) gene. This model develops a motor neuron disease and thus constitutes an accepted animal model for treating ALS.

[0139] Mice are treated at 75 days of age according to one or more of the following therapies: · Mice receive an i.p. injection with either 250 μg of an anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) or a control IgG anti body (IgG2a, #BE0089; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 after the trauma event and are examined after a further two-week intermission period; · Mice were subjected to i.p. injection with either 250 μg of anti-PD1 antibody (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg of anti-CTLA4 antibody (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.) or control IgG antibody (IgG2a, #BE0089, or polyclonal golden hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.) on days 1 and 4 of the experiment and were examined after a 2-week intermission period. · Mice were subjected to i.p. injection with GA weekly as described above after the traumatic event and were examined after a 2-week intermission period; · Mice were subjected to treatment with p300i or vehicle over the course of 1 week after the traumatic event and were examined 3 weeks later as described above; Some mice received an additional treatment period with an appropriate intermission period (about 3 weeks to 1 month).

[0140] The neurological function of movement is evaluated, for example, using a rotarod performance test (by which the ability of a mouse to stay on a rotating rod is evaluated), or a mouse grasps a vertical wire (2 mm in diameter) with a small loop at the lower end and is allowed to cling to it. The vertical wire enables the mouse to use both its front and hind legs to grasp the wire. The wire is maintained in a vertically oriented circular motion (with a radius of 10 cm) at 24 rpm. The time for which the mouse can cling to the wire is recorded by a timer.

[0141] ALS mice treated by one treatment period are expected to show significantly improved motor performance compared to a control group or an untreated group treated with IgG or vehicle. ALS mice that undergo two treatment courses and are examined after an appropriate interval are expected to show a long-lasting treatment effect. To maintain this treatment effect, the mice are subjected to effective treatment periods with appropriate intervals between each treatment period.

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Claims

1. A pharmaceutical composition comprising an active agent that causes a reduction in the level of systemic immunosuppression in an individual for use in treating a central nervous system (CNS) disease, disorder, condition or injury other than an autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RRMS), the pharmaceutical composition for administration according to a dosing schedule comprising at least two treatment courses (where each treatment course in turn comprises a treatment period followed by an intervening period).

2. The pharmaceutical composition according to claim 1, wherein the treatment period comprises administering the pharmaceutical composition to the individual, the treatment period being maintained until at least the level is lower than a reference standard, the administering being suspended during the intervening period, and the intervening period being maintained as long as the level is lower than the reference standard.

3. The reference standard is (a) the level of the systemic presence or activity of regulatory T cells measured in the most recent blood sample obtained from the individual prior to the administering; or (b) selected from the level of the systemic presence or activity of regulatory T cells or myeloid-derived suppressor cells characteristic of a population of individuals suffering from a CNS disease, disorder, condition or injury, the pharmaceutical composition according to claim 2.

4. The pharmaceutical composition according to claim 3, wherein the reference standard is the level of the systemic presence or activity of regulatory T cells measured in the most recent blood sample obtained from the individual prior to the administering.

5. The treatment period comprises administering the pharmaceutical composition to the individual, and the treatment period is maintained until at least the systemic presence or level of IFNγ-producing leukocytes increases beyond a reference standard, and the administering is suspended during the intervening period, and the intervening period is maintained as long as the level exceeds the reference standard, provided that the reference standard is (a) the level of the systemic presence or activity of IFNγ-producing leukocytes measured in the most recent blood sample obtained from the individual prior to the administering; or (b) selected from the level of the systemic presence or activity of IFNγ-producing leukocytes characteristic of a population of individuals suffering from a CNS disease, disorder, condition or injury, the pharmaceutical composition according to claim 1.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the treatment period is of a length between 1 week and 4 weeks.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the length of the intermittent period is between 2 weeks and 6 months.

8. The regulatory T cell is FoxP3 that expresses one or more of CD25, CD127, GITR, CTLA-4, or PD-1 ＋ cells, or FoxP3 that expresses one or more of the surface molecules of CD25, CD127, GITR, CTLA-4, or PD-1 － CD4 cells selected from ＋ The pharmaceutical composition according to any one of claims 1 to 7, which is a cell

9. The regulatory T cells are CD4 ＋ CD25 ＋ FoxP3 ＋ cells or CD4 ＋ CD25 ＋ FoxP3 － cells, and the pharmaceutical composition according to claim 8.

10. The active agent is (i) an antibody selected from the following: (a) anti-PD-1; (b) anti-PD-L1; (c) anti-PD-L2; (d) anti-CTLA-4; (e) anti-PD-1 in combination with interferon α; (f) anti-PD-1 in combination with anti-CTLA-4; (g) anti-CD47; (h) anti-OX40; (i) anti-VEGF-A; (j) anti-CD25; (k) anti-GITR; (l) anti-CCR4; (m) anti-TIM-3 / galectin 9; (n) anti-killer cell immunoglobulin-like receptor; (o) anti-LAG-3; or (p) anti-4-1BB; (ii) any combination of (a) to (p); (iii) any combination of (a) to (p) in combination with an adjuvant, for example, anti-CTLA-4 antibody in combination with anti-OX40 antibody and TLR9 ligand (such as CpG, etc.); (iv) a small molecule selected from the following: (a) p300 inhibitor, such as C646, C146, C375, and gemcitabine, etc.; (b) sunitinib; (c) polyoxometalate-1 (POM-1); (d) α,β-methylene adenosine 5'-diphosphate (APCP); (e) Arsenic trioxide (As ２ O ３ ); (f) GX15-070 (obatoclax); (g) retinoic acid antagonist, such as Ro41-5253 or LE-135, etc.; (h) SIRPα (CD47) antagonist, such as CV1-hIgG4, etc., as a single agent or in combination with anti-CD47 antibody; (i) CCR4 antagonist, such as AF399 / 420 / 18025, etc., as a single agent or in combination with anti-CCR4 antibody; (j) adenosine A2B receptor antagonist, such as PSB603, etc.; (k) antagonist of indoleamine-2,3-dioxygenase; or (l) HIF-1 regulator; (v) a protein selected from the following: (a) glycoprotein of the leaves of indosendan (NLGP); or (b) sCTLA-4; (vi) a silencing agent, such as miR-126 antisense and anti-galectin-1 (Gal-1), etc.; (vii) OK-432; (viii) combination of IL-12 and anti-CTLA-4; (ix) an antibiotic, such as vancomycin, etc.; or The pharmaceutical composition according to any one of claims 1 to 9, selected from any combination of (x) (i) to (ix).

11. The pharmaceutical composition according to claim 10, wherein the agent is an antibody specific for PD-1.

12. The pharmaceutical composition according to claim 10, wherein the agent is a p300 inhibitor.

13. The pharmaceutical composition according to claim 12, wherein the p300 inhibitor is C646.

14. A neurodegenerative disease, disorder or condition selected from Alzheimer's disease; amyotrophic lateral sclerosis; Parkinson's disease, Huntington's disease; primary progressive multiple sclerosis; secondary progressive multiple sclerosis; corticobasal degeneration; Rett syndrome; A retinal degenerative disorder selected from the group consisting of age-related macular degeneration and retinitis pigmentosa; anterior ischemic optic neuropathy; glaucoma; uveitis; depression; trauma-related stress or post-traumatic stress disorder; frontotemporal dementia; Lewy body dementia; mild cognitive impairment; posterior cortical atrophy; primary progressive aphasia; progressive supranuclear palsy or age-related cognitive impairment; the pharmaceutical composition according to any one of claims 1 to 13, for use in treating.

15. The pharmaceutical composition according to claim 14, wherein the neurodegenerative disease, disorder or condition is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease.

16. The pharmaceutical composition according to claim 15, for use in treating Alzheimer's disease.

17. The pharmaceutical composition according to any one of claims 1 to 13, for use in treating a CNS injury selected from spinal cord injury, closed head injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebral ischemia, optic nerve injury, myocardial infarction, organophosphate poisoning, and injury caused by tumor resection.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the treatment improves the motor and / or cognitive function of the CNS.

19. The pharmaceutical composition according to claim 18, for use in alleviating age-related loss of cognitive function.

20. The pharmaceutical composition according to claim 19, wherein the age-related loss of cognitive function occurs in an individual without a diagnosed disease.

21. The pharmaceutical composition according to claim 20, for use in alleviating loss of cognitive function resulting from acute stress or a traumatic episode.

22. The pharmaceutical composition according to any one of claims 18 to 21, wherein the cognitive function is learning, memory or both.

23. A method for treating a disease, disorder, condition or injury of the central nervous system (CNS) that does not include relapsing-remitting multiple sclerosis (RRMS), an autoimmune neuroinflammatory disease, the method comprising administering to an individual in need thereof a pharmaceutical composition according to any one of claims 1 to 24, wherein the pharmaceutical composition is administered according to a dosing schedule that includes at least two treatment courses, each treatment course in turn including a treatment period and a subsequent intermission period.

24. A pharmaceutical composition for use in treating a CNS disease, disorder, condition or injury that does not include RRMS of an autoimmune neuroinflammatory disease, the pharmaceutical composition comprising an active agent that causes a decrease in the level of systemic immunosuppression in an individual, selected from: (i) an antibody selected from: (a) anti-CD47; (b) anti-OX40; (c) anti-VEGF-A (bevacizumab); (d) anti-CD25; (e) anti-GITR (GITR-inducing mAb (DTA-1)); (f) anti-CCR4; (g) anti-TIM-3 / galectin 9; (h) anti-killer cell immunoglobulin-like receptor; (i) anti-LAG-3; or (j) anti-4-1BB; (ii) any combination of (a) to (j); (iii) any combination of (a) to (j) in combination with an adjuvant (e.g., a TLR9 ligand (e.g., CpG, etc.)); (iv) a protein selected from: (a) the glycoprotein of the leaves of indosendan (NLGP); or (b) sCTLA-4; (v) a small molecule selected from: (a) sunitinib; (b) polyoxometalate-1 (POM-1); (c) α,β-methylene adenosine 5'-diphosphate (APCP); (d) Arsenic trioxide (As ２ O ３ ); (e) GX15-070 (obatoclax); (f) a retinoic acid antagonist, e.g., Ro41-5253 or LE-135, etc.; (g) a SIRPα (CD47) antagonist, e.g., CV1-hIgG4, etc., as a single agent or in combination with an anti-CD47 antibody; (h) a CCR4 antagonist, e.g., AF399 / 420 / 18025, etc., as a single agent or in combination with an anti-CCR4 antibody; (i) an adenosine A2B receptor antagonist, e.g., PSB603, etc. (j) an antagonist of indoleamine-2,3-dioxygenase; or (k) an HIF-1 regulatory factor; (vi) a silencing agent, for example, miR-126 antisense and anti-galectin-1 (Gal-1), etc.; (vii) OK-432; (viii) a combination of IL-12 and anti-CTLA-4; (ix) an antibiotic, for example, vancomycin, etc.; or (x) any combination of (i) to (ix).

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