RU2815892C1 - Reduced systemic levels or activity of regulatory t-cells for treating disease and central nervous system involvement - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to neurology, and can be used to reduce systemic levels or activity of regulatory T-cells for treating diseases and CNS involvement. Method involves administering to an individual a pharmaceutical composition containing an anti-PD-1 antibody, an anti-PDL1 antibody or an anti-TIM-3 antibody. Pharmaceutical composition is administered using a dosing regimen comprising at least two courses of treatment, each therapeutic course includes a successive treatment session in which the pharmaceutical composition is administered to an individual, and a subsequent session without treatment, when the pharmaceutical composition is not administered to the individual. Introduction of the pharmaceutical composition during a treatment session is a single administration, and a session without treatment ranges from one month to three months.

EFFECT: use of the invention enables treating tauopathy by temporarily reducing the level of systemic immunosuppression in the circulatory system.

5 cl, 4 tbl, 107 dwg, 22 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[1] The present invention relates generally to methods and compositions for treating a disease, disorder, pathological condition or lesion of the central nervous system (CNS) by temporarily reducing the level of systemic immunosuppression in the circulatory system.

BACKGROUND OF THE INVENTION

[2] Most central nervous system (CNS) pathologies share a common neuroinflammatory component that is part of disease progression and contributes to disease spread. These pathologies include Alzheimer's disease (AD), an age-related neurodegenerative disease characterized by progressive loss of memory and cognitive function, in which the accumulation of beta-amyloid peptide (Aβ) aggregates is thought to play a key role in the cascade of CNS inflammation, leading to ultimately leading to neuronal destruction and tissue destruction (Akiyama et al., 2000; Hardy & Selkoe, 2002; Vom Berg et al., 2012). Despite the chronic neuroinflammatory response in neurodegenerative diseases, over the past decade, clinical and preclinical studies investigating immunosuppression-based therapies for neurodegenerative diseases have raised questions about the ineffectiveness of anti-inflammatory drugs (Breitner et al, 2009; Group et al, 2007; Wyss-Coray & Rogers, 2012). The inventors provide an innovative solution to overcome the shortcomings of existing methods of treating AD, as well as similar diseases and lesions of the central nervous system; this method is based on the inventors' particular understanding of the role of various components of the systemic and central immune system in maintaining the functioning and restoration of the central nervous system.

BRIEF DESCRIPTION OF THE INVENTION

[3] In one aspect, the present invention provides a pharmaceutical composition containing an active agent that causes a decrease in the level of systemic immunosuppression in a patient for use in the treatment of a disease, disorder, pathological condition or injury of the central nervous system that does not include an autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RPMC), wherein said pharmaceutical composition is intended to be administered through a dosage regimen comprising at least two courses of treatment comprising sequentially a course of treatment followed by a no-treatment period.

[4] In another aspect, the present invention provides a method of treating a disease, disorder, pathological condition or lesion of the CNS, which does not include the autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RPMC), said method comprising administering to a patient in need thereof , a pharmaceutical composition containing an active agent that causes a decrease in the level of systemic immunosuppression in accordance with the present invention, wherein said pharmaceutical composition is administered in a dosage regimen containing at least two courses of treatment, each course of treatment containing sequentially a course of treatment followed by a period without treatment.

BRIEF DESCRIPTION OF GRAPHIC MATERIAL

[5] In FIG. 1A-B depict choroid plexus (CP) activity during disease progression in 5XFAD AD-Tg transgenic mice. (A) mRNA expression levels of the icam1, vcam1, cxcl10, and ccl2 genes measured by real-time quantitative PCR (RT-qPCR) in choroid plexuses (CPs) collected from AD-Tg mice at 1, 2, 4, and 8 months of age. , presented as fold change compared to wild-type (WT) controls according to age (n=6-8 per group; Student's t test for each time point). (B) Representative microscopic images of CPs from 8-month-old AD-Tg mice and age-matched wild-type (WT) controls immunostained for epithelial tight junction molecules Claudin-1, Hoechst nuclear staining, and integrin ligand, ICAM-1 (scale bar, 50 µm). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[6] In FIG. Figures 2A-C show: (A) Quantification of ICAM-1 immunoreactivity in human CP (post-mortem) in young and elderly patients without CNS disorders and patients with AD (n=5 per group; one-way analysis of variance (ANOVA), followed by post hoc analysis (B) Newman-Keuls post-hoc analysis; (B) flow cytometry analysis of IFN-γ-expressing immune cells (intracellularly stained and pregated for CD45) in the choroid plexuses (CPs) of 8-month-old AD-Tg mice and wild-type controls by age group The shaded histogram represents isotype controls (n=4-6 per group; Student's t test) and (C) mRNA expression levels of the ifn-γ genes (IFN-γ) measured by RT-qPCR. in CP tissues isolated from 4- and 8-month-old AD-Tg mice compared with WT controls by age group (n=5-8 per group; Student's t test for each time point).In all panels, magnitude of error represents the mean ± standard error of the mean; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[7] In FIG. Figures 3A-B show (A) representative flow cytometry plots of the frequencies of CD4 ⁺ Foxp3 ⁺ splenocytes (pregated by TCRβ) in 8-month-old AD-Tg mice and WT control mice; and (B) quantification of splenocytes from 1-, 2-, 4-, and 8-month-old AD-Tg mice and control WT mice (n=6-8 per group; Student's t test for each time point). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[8] In FIG. Figure 4 shows the gating strategy and representative flow cytometry plots of splenocytes from AD-Tg/Foxp3-DTR ^+/- mice 1 day after the last DTx injection. DTx was injected intraperitoneally for 4 fixed days, achieving ~99% depletion of Foxp3 ⁺ cells.

[9] In FIG. 5A-G show the consequences of transient depletion of regulatory T cells in AD-Tg mice. (A) AD-Tg/Foxp3-DTR ⁺ (which express the DTR transgene) and non-DTR expressing AD-Tg littermates (AD-Tg/Foxp3-DTR ^- ) control group were treated with DTx for 4 designated days. The mRNA expression levels of the icam1, cxcl10 and ccl2 genes were measured by RT-qPCR (real-time quantitative PCR) in DTx-treated 6-month-old AD-Tg mice 1 day after the last DTx injection (n=6-8 per group; t -Student's t-test). (BD) Flow cytometry analysis of brain parenchyma (excluding the choroid plexus, which was excised separately) from 6-month-old AD-Tg mice and DTx-treated controls 3 weeks after the last DTx injection. Quantitative flow cytometry analysis showing increased numbers of D11b ^high /CD45 ^high mo-MF and CD4 ⁺ T cells (B), and representative flow cytometry plots (C) and quantitative analysis (D) of the frequency of CD4 ⁺ Foxp3 regulatory T cells ⁺ in the brain parenchyma of AD-Tg/Foxp3-DTR ⁺ mice and control AD-Tg/Foxp3-DTR treated with DTx (n=3-7 per group; Student's t test). (E) mRNA expression levels of foxp3 and il10 in the brain parenchyma of DTx-treated 6-month-old AD-Tg AD-Tg/Foxp3-DTR ⁺ mice and control AD-Tg/Foxp3- ^DTR mice 3 weeks after the last DTx injection (n=6-8 per group; Student's t-test). (F) Quantitative analysis of GFAP immunostaining demonstrating reduced astrogliosis in hippocampal slices from 6-month-old DTx-treated, AD-Tg/Foxp3-DTR ⁺ and AD-Tg/Foxp3-DTR ^- control mice 3 weeks after the last DTx injection (scale bar , 50 µm; n=3-5 per group; Student's t-test). (G) mRNA expression levels of il-12p40 and tnf-a in the brain parenchyma 3 weeks after the last DTx injection (n=6-8 per group; Student's t test). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[10] In FIG. 6A-E show the effect of transient depletion of regulatory T cells on Aβ plaques on learning and memory functions. (A) Representative microscopic images and (B) quantitative analysis of brains from DTx-treated 5-month-old AD-Tg/Foxp3-DTR+ mice and control AD-Tg/Foxp3-DTR- mice 3 weeks after the last DTx injection, immunostaining for Aβ -plaques and Hoechst nuclear staining (scale bar, 250 µm). The mean area and number of Aβ plaques in the hippocampal dentate gyrus (DG) and layer 5 cortex were quantified (in 6 μm brain sections; n=5 per group; Student's t test). In fig. Figures 6C-E show the results of Morris water maze (MWM) tests of DTx-treated 6-month-old AD-Tg/Foxp3-DTR+ mice and controls 3 weeks after the last DTx injection. After transient depletion of regulatory T cells, AD-Tg mice showed superior spatial learning and memory performance during the (C), (D) exploration, and (E) reversal phases of the WLM compared to AD-Tg controls (n=7- 9 per group; two-way repeated measures ANOVA followed by pairwise post hoc comparisons with Bonferroni test; ^* , P < 0.05 combined for recognition, probe, and reversal). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[11] In FIG. Figure 7 shows ifn-γ mRNA expression levels, measured by real-time quantitative PCR (RT-qPCR), in choroid plexuses (CPs) isolated from 6- and 12-month-old APP/PS1 AD-Tg mice (a mouse model of Alzheimer's disease). see materials and methods)), compared with wild-type controls by age group (n=5-8 per group; Student's t-test). The magnitude of the error represents the mean ± standard error of the mean; ^* , P<0.05.

[12] In FIG. 8A-I shows the therapeutic effect of weekly administration of glatiramer acetate (GA) to AD-Tg mice. (A) Schematic representation of the weekly GA treatment regimen. Mice (5 months old) were injected subcutaneously with GA (100 μg), twice during the first week (on days 1 and 4), and once a week thereafter, for a total duration of 4 weeks. Mice were tested for cognitive performance 1 week (Morris water maze, MWM), 1 month (radial water maze, Morris, RAWM), and 2 months (radial water maze, Morris, RAWM, using different experimental spatial settings) after the last injection. and inflammation of the hippocampus. In fig. 8B-D shows mRNA expression levels of genes in the hippocampus of untreated AD-Tg mice and AD-Tg mice treated with weekly GA at 6 months of age, showing (B) reduced expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-12p40, (C) increase in anti-inflammatory cytokines IL-10 and TGF-β and (D) neurotrophic factors, IGF-1 and BDNF, in mice treated weekly with GA (n=6-8 per group; Student's t test) . In fig. 8E-G AD-Tg mice (5 months of age) were treated weekly with either GA or vehicle (PBS) and compared with age-matched wild-type (WT) littermate controls at 6 months of age in the MWM test. Treated AD-Tg mice showed superior spatial learning and memory performance during the detection (E), exploration (F), and reversal (G) phases of MWM compared to controls (n=6-9 per group; two-way ANOVA with repeated measures ANOVA followed by pairwise post-hoc comparisons with Bonferroni test for comparisons of individual pairs; wild-type (WT) mice black circles; AD-Tg controls, white circles; AD-Tg treated, gray circles ). In fig. 8H-I presents the cognitive performance of the same mice in the RAWM test 1 month (H) or 2 months (I) after the last GA injection (n=6-9 per group; two-way repeated measures ANOVA followed by pairwise post hoc (“post-hoc”) comparisons with Bonferroni test for comparisons of individual pairs). Data are representative of at least three independent experiments. In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[13] In FIG. 9A-H show the additional therapeutic effects of weekly administration of GA to AD-Tg mice. In fig. A-B shows results in 5XFAD AD-Tg mice that received weekly GA or vehicle (PBS) treatment and were examined at the end of the first week of the administration regimen (after a total of two GA injections). Flow cytometry analysis of the frequency of CD4 ⁺ Foxp3 ⁺ splenocytes (A) and IFN-γ-expressing CP immune cells (B; intracellularly stained and pregated for CD45) in treated 6-month-old AD-Tg mice, compared with age-matched WT mice (n=4-6 per group; one-way ANOVA followed by Newman-Keuls post-hoc analysis). (C) mRNA expression levels of the icam1, cxcl10, and ccl2 genes measured by RT-qPCR in the CPs of 4-month-old AD-Tg mice treated weekly with both GA and vehicle and examined at both the end of 1 and 4 week 1 of the weekly GA regimen (n=6-8 per group; one-way ANOVA followed by Newman-Keuls post-hoc analysis). In fig. Figures 9D-E show representative images of brain sections from 6-month-old AD-Tg/CX3CR1 ^GFP/+ bone marrow (BM) chimeras after 1 week of GA treatment. CX3CR1 ^GFP cells were localized to the CP of the third ventricle (3V; i), the adjacent ventricular space (ii), and the CP of the lateral ventricles (LV; iii) in AD-Tg mice treated weekly with GA (D; scale bar, 25 μm ). Representative orthogonal z-axis projections of confocal images (stacks) showing colocalization of GFP+ cells with the myeloid marker, CD68, in the CP of 7-month-old AD-Tg/CX3CR1GFP/+ mice treated with weekly GA, but not in control AD-Tg/CX3CR1 mice ^GFP/+ treated with PBS (E, scale bar, 25 μm). (F) CX3CR1 ^GFP cells colocalize with the myeloid marker IBA-1 in the brains of GA-treated AD-Tg/CX3CR1 ^GFP/+ mice, adjacent to Aβ plaques and coexpressing the myeloid marker, IBA-1 (scale bar, 25 μm). In fig. 9G-H shows representative flow cytometry plots of cells isolated from the hippocampus of 4-month-old untreated AD-Tg WT mice and AD-Tg mice in the second week of a weekly GA regimen. CD11b ^high /CD45 ^high mo-MFs were gated (G) and quantified (H; n=4-5 per group; one-way ANOVA followed by Newman-Keuls post-hoc analysis). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[14] In FIG. 10A-H depict the therapeutic effect of administering p300 inhibitor (C646) to AD-Tg mice. In fig. 10A-B mice, 18 months of age, were treated with both p300i and vehicle (DMSO) for 1 week and examined the day after cessation of treatment. Representative flow cytometry plots show an increase in the frequency of IFN-γ-expressing CD4 ⁺ T cells in the spleen (A) and the number of IFN-γ-expressing cells in the CP (B) following p300i treatment. In fig. 10C-E show representative microscopic images (C) and quantitative analysis of Aβ plaque volume in the brains of 10-month-old AD-Tg mice that received p300i or vehicle (DMSO) for 1 week and were then examined an additional 3 weeks later. Brain preparations were immunostained for Aβ plaques and nuclei were stained with Hoechst (n=5 per group; scale bar, 250 μm). The average area of Aβ plaques and the number of plaques in the dentate gyrus of the hippocampus (DG) (D) and in the 5th layer of the cerebral cortex (E) were calculated (in 6 μm brain sections; n = 5-6 per group; t- Student's t test). (F) Schematic representation of the treatment regimen of administering p300i (or DMSO as vehicle) to different groups of AD-Tg mice at 7 months of age, for 1 or 2 sessions. In fig. 10G-H shows the change in the average percentage coverage of the cerebral cortex by Aβ plaques (5th layer) (G) and the change in the average levels of cerebral soluble protein Aβ _1-40 and Aβ _1-42 (H), relative to the untreated AD group -Tg (mean levels of AB _1-40 and Aβ _1-42 in the untreated group, 90.5 ± 11.2 and 63.8 ± 6.8 pg/mg total, respectively; n = 5-6 per group; one-way analysis of variance ANOVA, followed by post-hoc Newman-Keuls analysis). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[15] As shown in FIG. 11A-D, PD-1 blockade increases the percentage of IFN-γ-producing CD4 ⁺ T cells in the spleen as well as IFN-γ expression in the choroid plexus in AD-Tg mice. 10-month-old AD-Tg mice were injected intraperitoneally on days 1 and 4 with 250 μg of anti-PD-1 or control IgG, and the effect on systemic immune response and CP activity was examined on days 7–10. (A-B) Representative flow cytometry plots (A) and quantitative analysis (B) of the frequency of CD4+IFN-γ+ splenocytes (intracellularly stained and pregated for CD45 and TCR-β) in AD-Tg mice treated with anti -PD-1 or IgG, and untreated AD-Tg mice and WT controls (n=4-6 per group; one-way ANOVA followed by Newman-Keuls post-hoc analysis; ^** , P <0.01 between designated treatment groups; error bars represent mean ± SEM). (C) ifn-g mRNA expression levels measured by RT-qPCR in the CP of anti-PD-1-treated AD-Tg mice compared with IgG-treated and with untreated AD-Tg controls (D) Gene Ontology Terms (GO Terms) RNA-Seq enrichment in CPs of the same mice (n=3-5 per group; one-way ANOVA followed by Newman-Keuls post-hoc analysis ; ^* , P<0.05) (gray scale corresponds to the negative decimal logarithm of the P value).

[16] As shown in FIG. 12A-B, PD-1 blockade reduces cognitive decline in AD-Tg mice. 10-month-old AD-Tg mice were intraperitoneally injected on days 1 and 4 with 250 μg of both anti-PD-1 and control IgG, and the effect on pathology was examined 1 or 2 months later, (A) demonstrating the effects of mouse training AD-Tg in the RAWM test after 1 session of treatment with anti-PD-1 or control IgG, and (B) demonstrating the effect of a single session of anti-PD-1 treatment, or 2 sessions with a 1-month training interval. Single arrows indicate treatment time points, double arrows indicate cognitive testing time points. Cognitive performance of mice treated with anti-PD-1 and IgG, compared with age-matched wild-type AD-Tg mice and untreated mice, was assessed by the average number of errors per day on learning and memory tasks in the RAWM test (n=6- 8 per group; two-way repeated measures ANOVA followed by pairwise post-hoc comparisons with Bonferroni test).

[17] In FIG. 13A-D are representative microscopic images showing that PD-1 blockade reduces AD pathology (A) and quantitative analysis (B, C, D) of Aβ plaque load and astrogliosis in the brains of AD-Tg mice treated at age 10 months, both anti-PD-1 (in 1 or 2 sessions, as shown in Fig. 12A-b) and IgG, and then tested at 12 months of age. Brain preparations were immunostained for Aβ plaques (red), GFAP (a marker of astrogliosis, green), and nuclei stained with Hoechst (n=4–5 per group; scale bar, 50 μm). The average area and number of Aβ plaques in the hippocampal dentate gyrus (DG) and layer 5 cortex were quantified, and GFAP immunoactivity in the hippocampus was measured (in 6 μm brain sections; n=5-6 per group; t- Student's t test). In all panels, error values represent the mean ± SEM; ^* , P<0.05; ^** , P<0.01; ^*** , P<0.001.

[18] In FIG. 14 presents the effect of different dosage and frequency of administration of anti-PD-1 antibody on cognitive decline in AD-Tg mice, and illustrates the dosing schedule and the effect of anti-PD-1 antibody treatment on spatial learning and memory using the radial arm water test. labyrinth (RAWM) at the age of 7 months. Black arrows indicate treatment time points, illustrations indicate cognitive testing time points.

[19] In FIG. 15A-H show the effect of repeated administration of anti-PD-1 antibody on cognitive decline in AD-Tg mice. 5XFAD mice were treated with both PD-1 specific antibody and control IgG starting at 3 months of age; treatment was carried out once a month until 5 months of age (three injections in total). The experimental design is shown in (A). Black arrows indicate treatment time points, and illustrations show cognitive testing time points. (B) RAWM training at 5 months of age, anti-PD-1-treated 5XFAD mice (n=7), 5XFAD mice (n=9) treated with control antibody (IgG); and wild-type (WT) mice (n=8). (C) RAWM training at 6 months of age, anti-PD-1 treated 5XFAD mice (n=7), 5XFAD mice (n=9) treated with control antibody (IgG); and wild-type (WT) control mice (n=8). (two-way repeated measures ANOVA with Dunnett's post hoc test for multiple comparisons between the two 5XFAD treatment groups). (D) Comparison of learning outcomes between anti-PD-1 and IgG treated groups at 5 and 6 months of age; values indicating the number of errors for each mouse are taken from the last measurement taken on the second day of testing. (E) Representative immunofluorescence images and (F) quantitative analysis of Neu-N+ neurons in the hippocampal base of 5XFAD mice treated with anti-PD-1 (n=9); IgG-treated 5XFAD mice (n=10) and control wild-type (WT) mice (n=6). (G) Representative immunofluorescence images of hippocampal neurons showing increased caspase-3 activity in IgG-treated 5XFAD mice (n=8) compared to anti-PD-1-treated 5XFAD mice (n=5). (H) Quantification of activated caspase-3-immunoreactive Neu-N+ cells in the CA3 region of the hippocampus (one-way ANOVA and Fisher's exact test). Scale bars, 100 µm (e, g). Data are presented as mean ± standard error of the mean; ^* P<0.05, ^** P<0.01, ^*** P<0.001

[20] In FIG. 16 presents the effect of a single administration of anti-TIM-3 antibody on cognitive decline in AD-Tg mice, and illustrates the dosing schedule and effect of anti-TIM-3 antibody treatment on spatial learning and memory using the radial arm water maze (RAWM) test. at the age of 7 months. Black arrows indicate treatment time points, illustrations indicate cognitive testing time points.

[21] As shown in FIG. 17A-J, PD-1 blockade attenuates cognitive decline in AD-Tg mice. 5XFAD mice (7 months old) were treated with both PD-1-specific antibody, PD-L1-specific antibody, and an isotype-matched control antibody (IgG). The experimental design is shown in (A). Black arrows indicate treatment time points, and illustrations show time points of cognitive assessment using RAWM. (B) Training of 5XFAD mice in the RAWM test (males and females used in equal proportions in all groups, including the IgG control), treated as 0.1 mg/mouse (n=8), 0.5 mg/mouse (n =9), and 1.5 mg/mouse (n=9) injections of a single dose of anti-PD-L1-specific antibody, or control IgG at 1.5 mg/mouse (n=10). Age-matched wild-type (WT) littermate mice were used as an additional control group (n=10). (C) Comparison of learning in the RAWM test of 5XFAD mice treated with both 0.5 mg anti-PD-1-specific antibody (n=14) and 0.5 mg anti-PD-L1-specific antibody (n=7) ; and also tested IgG control antibody (n=15) and WT controls (n=19) (two-way repeated measures ANOVA with Dunnett's post hoc test for multiple comparisons between each anti-PD-1 treated group and IgG treated group ). The combined results of two experiments are presented. (D) Representative immunofluorescence images of brain preparations immunostained for Aβ (red), GFAP (green), and DAPI nuclear staining (scale bars, 100 μm), and quantification (E, F) of Aβ in 5XFAD mice treated with anti-PD -1 (n=9), anti-PD-L1-treated 5XFAD mice (n=10), and IgG-treated 5XFAD mice (n=9). (G) GFAP in anti-PD-1-treated 5XFAD mice (n=8), anti-PD-L1-treated (n=10), IgG-treated 5XFAD mice (n=15), and WT mice (n=6) , as assessed 1 month after treatment. The mean area and number of plaques (in 6-μm brain sections) in the dentate gyrus (DG) and cerebral cortex (layer V) were quantified, and GFAP immunoactivity in the hippocampus was measured (one-way ANOVA and Fisher's exact test). (H) Representative immunofluorescence images and (I) quantification of synaptophysin, assessed 1 month after treatment, in brain preparations from anti-PD-1-treated 5XFAD mice (n=6), anti-PD-L1-treated 5XFAD mice ( n=8) and IgG-treated 5XFAD mice (n=8). Synaptophysin immunoreactivity was measured in the DG and CA3 regions of the hippocampus (one-way ANOVA and Fisher's exact test). (J) Levels of il-12p40 mRNA expression relative to il-10 expression measured by RT-gPCR in hippocampal tissue isolated from 5XFAD mice 1 month after treatment with control IgG (n=5), anti-PD-1 (n=5) , or anti-PD-L1 (n=5) (one-way ANOVA and Fisher's exact test). (B-C, EG, IJ) Data are presented as mean ± SEM; ^* P<0.05, ^** P<0.01, ^*** P<0.001

[22] In FIG. 18A-B show that PD-L1 expression increases in the CP with age, with (A) showing PDL1 expression in the CP of young (left column) and adult (right column) mice as measured by RT-gPCR; and (B) immunohistochemical staining of epithelial PD-L1 expression in the CP of young (left micrograph) and adult (right micrograph) mice is shown. LV; lateral ventricle.

[23] In FIG. 19A-B are graphs of outer nuclear layer (ONL) thickness across the entire retina measured in individual eyes of RCS rats that received intraperitoneal injection of anti-PD1 mAb (n=10 rats; 20 eyes), IgG (n=10 rats, 20 eyes); , or untreated animals (n=4 rats, 8 eyes) by histological analysis based on hematoxylin and eosin (H&E) staining, (A) shows all animals and (B) shows only responders. Data are presented as mean ± standard error of the mean; ^* P<0.05, ^** P<0.01, ^*** P<0.001

[24] In FIG. 20A-B are plots of outer nuclear layer (ONL) thickness across the entire retina measured in individual eyes of RCS rats that received intravitreal injection of anti-PD1 mAb (n=6) or IgG (n=5), or by histological analysis based on hematoxylin and eosin (H&E) staining, showing both eyes (the injected eye and the contralateral eye that was not injected); (A) shows animals treated with anti-PD1 mAb and (B) animals treated with IgG. Data are presented as mean ± standard error; significant differences were determined using Student's t test for each individual sample point and are indicated by asterisks ( ^* P < 0.05).

[25] As shown in FIG. 21A-E, Brainstem PD-1/PD-L1 blockade in DM-hTau mice increases monocyte recruitment to the brain. 10-month-old DM-hTAU mice treated with 0.5 mg anti-PD-L1 (n=10 ) or IgG-matched control antibody (IgG) (n=17), and littermates of WT mice by age group (n=13) were analyzed 14 days after treatment using flow cytometry. (A) Splenocyte gating strategy by flow cytometry and (B) quantitative analysis of regulatory FoxP3 ⁺ T cells, and (C) memory effector T cells (T _EF ), CD44 ⁺ CD62L ^-/low (T _EF ), and (C ) effector T cells (T _EF ) memory CD44 ⁺ CD62L ^-/low (T _EF ) compared with central T cells (T _CM ) memory CD44 ⁺ CD62L ^high . (D) Flow cytometry gating strategy for CD45 ^high CD11b ⁺ and CD45 ^high CD11b ⁺ brain myeloid cells. (E) Brains from the same mice were dissected and analyzed for the presence of infiltrating CD45 ^high CD11b ⁺ myeloid cells. Quantitative analysis of brain CD45 ^high CD11b ⁺ cells showing increased frequency of infiltrating myeloid cells 14 days after PD-L1 blockade (n=10) compared with IgG-treated DM-hTAU mice (n=16) and WT littermates ( n=13). The results of two independent experiments were combined. Data are shown as mean ± SEM; ^* P<0.05, ^** P<0.01, ^*** P<0.001

[26] As shown in FIG. 22A-G, Blockade of brainstem PD-1/PD-L1 signaling in DM-hTau mice increases monocyte recruitment to the brain. Male mice expressing the human tau gene with two mutations (K257T/P301S, double mutant, DM-hTAU) (group mean age, 8 months) were treated with an anti-PD-1-specific antibody, anti-PD-L1-specific antibody or control antibody of the corresponding isotype (IgG) (one intraperitoneal injection of 0.5 mg/mouse); the experimental design is presented in (A). The black arrow shows the treatment time point, and the illustrations show the cognitive assessment time points. (B) Effect of PD-1/PD-L1 blockade on spatial memory using the T-maze. DM-hTAU mice treated with both anti-PD-1 (n=10) and anti-PD-L1 (n=10) showed a preference for the new arm compared to IgG controls (n=16) (B-C ). Littermate WT mice by age group (n=19) were used as an additional control group. The results of two independent experiments were combined. (C) Representative heat map plots of the time three groups of subjects spent in different arms. (D) Performance of the Y-maze task in DM-hTAU mice treated with anti-PD-1 (n=6), anti-PD-L1 (n=4), or IgG isotype control (n=6), and also wild-type mice of the same litter (n=5). (E) Representative immunofluorescence images and (F) quantitative analysis of GFAP immunoreactivity, assessed 1 month after treatment, in the hippocampus of DM-TAU+IgG (n=6), DM-hTAU+anti-PD-1 (n= 6), DM-hTAU + anti-PD-L1 (n=4), and untreated WT littermate mice (n=5). Immunoreactivity to GFAP in the brain of DM-hTAU + IgG (n=11), DM-TAU + anti-PD-1 (n=11), DM-tau + anti-PD-L1 (n=4) and untreated mice littermate WT mice (n=5), compared with IgG-treated controls (one-way ANOVA and Fisher's exact test). Scale bars, 100 μm (G) tnf-a mRNA expression levels; il-6 and il-12p40 versus il-10 expression measured by RT-gPCR in hippocampus isolated from DM-hTAU mice 1 month after treatment with control IgG (n=5), anti-PD-1 (n= 6), or anti-PD-L1 (n=4) (one-way ANOVA and Fisher's exact test), (b, d, fg) Data are presented as mean ± standard error of the mean; ^* P<0.05, ^** P<0.01, ^*** P<0.001

[27] In FIG. 23A-G shows that blockade of the PD-1/PD-L1 signaling pathway reduces hyperphosphorylation in DM-tau mice. Immunostaining of neurofibrillary tangles (NFTs) in the brains of 8-month-old DM-hTAU mice 1 month after treatment with anti-PD-1, anti-PD-L1, or isotype-matched control antibody, or isotype-matched control antibody (IgG) (AF). (A, D) Representative immunofluorescence images and (B, C, E, F) quantitative analysis of AT-100 and AT-180. AT-100 and AT-180 immunoreactivity was measured in hippocampal regions CA1 and CA3 in DM-hTAU + IgG (n=11), DM-hTAU + anti-PD-1 (n=10), DM-tau + anti-PD mice -L1 (n=4) (one-way ANOVA and Fisher's exact test). Scale bars, 100 μm (B-C, E-F) Data are presented as mean ± SEM. (G) Male and female mice (equally distributed among all test groups) expressing human tau protein with two mutations (K257T/P301S, double mutant, DM-hTAU) (mean age 9 months) were treated with both single-dose injections of anti- PD-L1-specific antibody 0.1 mg/mouse (n=7), 0.5 mg/mouse (n=10) or 1.5 mg/mouse (n=9), and control IgG 1.5 mg /mouse (n=10). The effect of PD-L1 blockade on spatial memory was determined using the T-maze. Littermate WT mice by age group were used as an additional control group (n=10) (one-way ANOVA and Fisher's exact test); *P<0.05, ^** P<0.01, ^*** P<0.001.

[28] In FIG. 24A-B show how PD-1 blockade enhances neurogenesis in the hippocampus of 5XFAD mice, showing (A) parasagittal brain sections immunostained for the neuronal marker NeuN (green), DCX (red), and nuclear staining Hoechst (blue); and (B) is a graph quantifying staining in anti-PD-1-treated animals, IgG immune controls, and age-matched wild-type controls.

[29] In FIG. 25A-B show how PD-1 blockade enhances hippocampal synaptic plasticity in 5XFAD mice, with (A) showing parasagittal brain sections immunostained for VgluT1 (red); and (B) is a graph quantifying staining in anti-PD-1-treated animals, IgG immune controls, and age-matched wild-type controls.

[30] In FIG. 26A-B show how PD-1 blockade reduces neuronal loss in the subiculum in 5XFAD mice, with (A) showing parasagittal brain sections immunostained for the neuronal marker NeuN (green); and (B) is a graph quantifying staining in anti-PD-1-treated animals, IgG immune controls, and age-matched wild-type controls.

DETAILED DESCRIPTION OF THE INVENTION

[31] Immune checkpoint mechanisms, which involve cell-intrinsic down-regulation of activated T cell responsiveness and effector function through inhibitory receptors, support the systemic immune system. homeostasis and autoimmune tolerance (Joller et al, 2012; Pardoll, 2012). In recent years, blockade of these immune checkpoints, such as the programmed cell death-1 (PD-1) signaling pathway (Francisco et al, 2010), has demonstrated remarkable antitumor efficacy, highlighting the potential to unleash the power of the immune system in the fight against various malignancies. It was recently shown (WO 2015/136541, Baruch et al., 2016) that administration of anti-PD-1 antibodies (anti-PD-1 antibodies) to an animal model of Alzheimer's disease leads to clearance of Aβ, reversal of cognitive decline, and is associated with decreased neuroinflammatory response. Thus, systemic immunosuppression interferes with the ability to clear AD pathology, and by removing restrictions on systemic immunity, AD pathology can be mitigated.

[32] Without wishing to be bound by theory, the authors propose that immune checkpoint blockade activates a cascade of immunological events that begins in the periphery and culminates in multiple activities within the brain. Initially, the immune response increases the availability of IFN-γ in secondary lymphoid organs (lymph nodes, spleen, etc.) and circulating monocytes in the periphery. This immune response results in immune activation of the choroid plexus (CP), an epithelial layer in the ventricles of the brain that forms the blood-CSF-B barrier and serves as a selective gateway for leukocytes entering the CNS. The effect of inhibitory immune checkpoint blockade on leukocyte CP gateway activity is mediated by IFN-γ-induced expression of leukocyte migration molecules (adhesion molecules and chemokines) through the CP epithelium, which mediates leukocyte migration. This increased expression results in the recruitment of monocyte-derived macrophages and immunoregulatory cells to lesions within the brain. Importantly, this recruitment leads to complex effects on brain function, including reduction of plaque volume, restoration of immunological balance, elimination of local inflammation, reduction of gliosis, reduction of synaptic loss, increased neurogenesis, increased protection of neurons and increased survival of neurons, which together leads to neuroprotection and/or attenuation of cognitive decline.

[33] Immune checkpoints are immune system molecules that either turn on a signal (costimulatory molecules) or turn off a signal. The four stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily—CD27, CD40, OX40, GITR, and CD137. Two other checkpoint stimulatory molecules belong to the B7-CD28 superfamily, CD28 itself, and ICOS. Many checkpoint inhibitory molecules are known, including, but not limited to, A2aR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG-3, PD-1, TIM-3 and VISTA.

[34] The present invention provides a method of treating a disease, disorder, condition or lesion of the central nervous system (CNS). In one embodiment, the disclosed method of treating a disease, disorder, condition, or lesion of the central nervous system (CNS) does not include the autoimmune neuroinflammatory disease relapsing-remitting multiple sclerosis (RRMS). The disclosed method comprises administering to a patient in need thereof an active agent that causes a decrease in the level of systemic immunosuppression, wherein said active agent is administered in a dosage regimen containing at least two courses of treatment, each course of treatment comprising sequentially a treatment session followed by there should be a break without treatment.

[35] In another aspect, the present invention is directed to an active agent that causes a decrease in the level of systemic immunosuppression in a patient, or a pharmaceutical composition containing an active agent, for use in the treatment of a disease, disorder, pathological condition or lesion of the central nervous system, other than an autoimmune neuroinflammatory disease, relapsing-remitting multiple sclerosis (RRMS), where the specified pharmaceutical composition is administered in a dosage regimen containing at least two courses of treatment, with each course of treatment containing sequentially a treatment session followed by a break without treatment.

[36] In some embodiments, the dosing regimen is calibrated such that the level of systemic immunosuppression is temporarily reduced.

[37] As used herein, the term “treatment” refers to the means of obtaining the desired physiological effect. The effect may be therapeutic in terms of partial or complete cure of the disease and/or symptoms associated with the disease. The term refers to inhibition of disease, i.e. stopping or slowing down its development; or mitigation of the disease, i.e. achieving disease regression.

[38] As used herein, the term “systemic presence” of regulatory or effector T cells refers to the presence of regulatory or effector T cells (as measured by their level of activity) in the circulatory immune system, that is, in the blood, spleen and lymph nodes. It is well known in the field of immunology that the cell population profile in the spleen is reflected in the cell population profile in the blood (Zhao et al, 2007).

[39] The present treatment is applicable to both patients who demonstrate increases in systemic immunosuppression and those who do not. Sometimes a subject who requires treatment in accordance with the present invention has a certain level of peripheral immunosuppression, which is reflected by an increased frequency or number of regulatory T cells in the circulating blood, and/or their increased functional activity, and/or a decrease in the number of leukocytes producing IFNγ, and/or decreased leukocyte proliferation in response to stimulation. Increases in the frequency or number of regulatory T cells can be expressed as total numbers or as a percentage of total CD4 cells. For example, in accordance with the present invention, it has been found that an animal model of Alzheimer's disease has a higher frequency of Foxp3 from CD4 cells compared to wild-type mice. However, even if the levels of systemic regulatory T cells are not increased, their functional activity is not enhanced, the level of IFNγ-producing leukocytes is not reduced, or the proliferation of leukocytes in response to stimulation is not reduced in the subject, the method proposed by the present invention reduces the level or systemic immunosuppressive activity, is effective for the treatment of diseases, disorders, pathological conditions or lesions of the central nervous system, other than the autoimmune neuroinflammatory disease RPMS. Importantly, this systemic immunosuppression may also involve additional types of immune cells other than regulatory T cells, such as myeloid-derived suppressor cells (MDSCs) (Gabrilovich & Nagaraj, 2009).

[40] The level of systemic immunosuppression can be detected by various methods, which are well known to one skilled in the art. For example, the level of regulatory T cells can be measured by flow cytometry of peripheral blood mononuclear cells or T lymphocytes immunostained for both cell surface and nuclear intracellular markers Treg (Chen & Oppenheim, 2011), CD45, TCR-β, or CD4 lymphocyte markers, by measuring the amount of antibodies specifically bound to the cells. The functional activity of regulatory T cells can be measured in a variety of ways; for example, the thymidine incorporation assay is widely used, in which suppression of proliferation of CD4 ⁺ CD25 ^- T cells (conventional T cells) stimulated by anti-CD3 mAb is measured by incorporation of [ ³ H]thymidine or application of CFSE (5-(and 6)- carboxyfluorescein diacetate succinimidyl ester, which is capable of penetrating cells; cell division is measured as a progressive decrease in half of CFSE fluorescence intensity). The number of leukocytes producing IFNγ, their activity or ability to proliferate can be easily assessed by those skilled in the art using common methods; for example, the level of IFNγ-producing leukocytes can be measured by flow cytometry of peripheral blood mononuclear cells, after brief ex-vivo stimulation, and GolgiStop, and immunostaining with IFNγ-intracellular dye (for example, using the BD Biosciences Cytofix/cytoperm™ fixation/permeabilization kit ), by collecting the conditioned medium of these cells and quantifying the level of secreted cytokines, using ELISA, or by comparing the ratio of various cytokines in the conditioned medium, for example IL2/IL10, IL2/IL4, INFγ/TGFβ, etc. MDSC levels in human peripheral blood can be readily determined by one skilled in the art, for example, by flow cytometric analysis of the frequency of DR ^- /LIN ^- /CD11b+, DR ^- /LIN ^- /CD15+, DR ^- /LIN ^- /CD33+ and DR(-/low)/CD14+ as described (Kotsakis et al, 2012).

[41] In humans, peripheral/systemic immunosuppression may be considered elevated when the total number of regulatory T cells in the circulatory system is greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or greater than in a healthy control population, the proportion of regulatory T cells to total CD4+ cells is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or higher than in the healthy control population, or functional Regulatory T cell activity is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or higher than in a healthy control population. In addition, peripheral/systemic immunosuppression may be considered elevated when the level or activity of IFNγ-producing lymphocytes is 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% less than in a healthy control population. ; or leukocyte proliferation in response to stimulation is reduced compared to a healthy control population by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

[42] An agent may be considered an agent that causes a decrease in the level of systemic immunosuppression when, when the agent is administered to a patient, the total number of regulatory T cells in the circulatory system of that subject is reduced by 10, 20, 30, 40, 50, 60, 70, 80. 90 or 100% compared to the level before administration of the agent, the proportion of regulatory T cells in the total number of CD4+ cells is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% compared to the healthy control population , or the functional activity of regulatory T cells is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% compared to the level before administration of the agent. In addition, an agent may be considered to cause a decrease in the level of systemic immunosuppression when, upon administration of the agent to a patient, the total number of IFNγ-producing leukocytes or their activity increases by 10, 20, 30, 40, 50, 60, 70, 80, 90. 100% or more; or leukocyte proliferation in response to stimulation increases relative to a healthy control population by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or more.

[43] In some embodiments, the active agent causes a decrease in the level of systemic immunosuppression by removing restrictions imposed on the immune system by one or more immune checkpoints, for example, blockade of one or more immune checkpoints.

[44] In some embodiments, a decrease in the level of systemic immunosuppression is associated with an increase in the systemic presence or activity of IFNγ-producing leukocytes.

[45] In some embodiments, the active agent causes a decrease in the level of systemic immunosuppression and therefore an increase in the systemic presence or activity of effector T cells.

[46] In some embodiments, a decrease in the level of systemic immunosuppression is associated with an increase in the systemic presence or activity of the cytokine IFNγ.

[47] In some embodiments, a decrease in the level of systemic immunosuppression is associated with a decrease in the systemic presence or activity of regulatory T cells.

[48] In some embodiments, the decrease in the level of systemic immunosuppression is associated with a decrease in the systemic presence or activity of the cytokine IL-10.

[49] In some embodiments, the decrease in the level of systemic immunosuppression is associated with a decrease in the systemic presence or activity of myeloid-derived suppressor cells (MDSCs).

[50] In some embodiments, the active agent causes a decrease in the level of systemic immunosuppression and therefore an increase in the systemic presence or activity of effector T cells.

[51] Checkpoints that can be manipulated to release systemic immunosuppression, referred to here as a pair consisting of an immune checkpoint receptor and its native ligand, or either of the two partners. For example, PD-1, which has two known ligands, is called "PDL1" or "PDL2", while B7H3, which has not yet been identified, is simply called "B7H3". Checkpoints that can be manipulated to release systemic immunosuppression according to the present invention include, but are not limited to, PD-1-PDL1, PD-1-PDL2, CD28-CD80, CD28-CD86, CTLA-4-CD80, CTLA-4 -CD86, IKO-B7RP1, B7H3, B7H4, B7H7, B7-CD28-like molecules, BTLA-HVEM, KIR-MHC class I or II, LAG3-MHC class I or II, CD137-CD137L, OX40-OX40L, CD27- CD70, CD40L-CD40, TIM3-GAL9, V-domain immunoglobulin suppressor of T-cell activation (VISTA), stimulator of interferon genes (STING), T-cell immunoglobulin and tyrosine-based immunoglobulin inhibitory motif (TIGIT) glucocorticoid-induced TNF- related protein (GITR), A2aR-adenosine and indoleamine 2,3-dioxygenase (IDO)-L-tryptophan.

[52] Agents capable of blocking immune checkpoints are known in the art (Colombo & Piconese, 2007) and can be used in accordance with the present invention. Each of the following publications, and Pardoll, 2012, are incorporated by reference as if the text were fully reproduced herein.

[53] In some embodiments, an active agent that can be used in the present invention can be an antibody. The antibody disclosed herein may be a polyclonal antibody, a monoclonal antibody, a dimer, a multimer, a multispecific antibody, a human antibody, a humanized antibody, a recombinant antibody, a chimeric antibody, a bifunctional antibody, a cell-associated Ig receptor-like antibody, a linear antibody, a diabody, a minibody or nanobody, provided that the fragment exhibits the required biological activity, and their single-chain derivatives. The antibody may be a full-length immunoglobulin molecule containing the VH and VL domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3, or an immunologically active fragment of a full-length immunoglobulin molecule, such as a single domain antibody (sdAb), single chain variable fragment (scFv), Fab fragment, F(ab')2 fragment, Fc fragment, Fd fragment, Fv fragment. The antibody may be obtained from any vertebrate species (e.g., human, goat, horse, donkey, mouse, rat, rabbit, or chicken) and may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgA, IgD, IgE, IgG and IgM) or subclass (IgG1, IgG2, IgG3, IgG4, IgG1 and IgG2). Functionally, the antibody disclosed herein may be an antagonist antibody, that is, an antibody that inhibits a biological activity, or the antibody described herein may be an agonist antibody, that is, an antibody that promotes a biological activity. Likewise, an antibody described herein may be a neutralizing antibody, that is, an antibody that can block or neutralize a biological activity. For general requirements for the disclosure of information about the structure of natural antibodies, non-naturally occurring antibodies and their antigen-binding fragments, see, for example: Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995), both sources are incorporated herein by reference in their entirety.

[54] The antibody described herein may be, without limitation, anti-PD-1, anti-PDL1, anti-PDL2, anti-CTLA-4, anti-CD80, anti-CD86, anti-B7RP1, anti-B7- H3, anti-B7-H4, anti-B7-H7, anti-BTLA, anti-HVEM, anti-CD-27, anti-CD40, anti-CD40L, anti-CD70, anti-CD80, anti-CD86, anti- CD137, anti-CD137L, anti-OX40, anti-OX40L, anti-TIM-3, anti-Galectin9, anti-KIR, anti-LAG-3, anti-ICOS, anti-VISTA, anti-STING, anti-TIGIT, anti-GITR or any combination thereof. The antibody described herein can be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0 .1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg/kg - 15 mg/kg, 0.3 mg/ kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg/kg - 10 mg/kg, 1.5 mg/ kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[55] Programmed death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a cell surface receptor belonging to the immunoglobulin superfamily and expressed on T cells and pro-B cells. PD-1 binds two ligands, PDL1 and PDL2. Acting as an immune checkpoint, PD-1 plays an important role in down-regulating the immune system by preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effects of PD-1 occur through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while reducing apoptosis in regulatory T cells (suppressor T cells). Essentially, compounds that inhibit PD-1 function, such as PD-1 inhibitors, PDL1 inhibitors and/or PDL2 inhibitors, serve to activate the immune system. One class of PD-1 inhibitors includes antagonistic or neutralizing antibodies to PD-1, PDL1, and PDL2. Many antagonistic or neutralizing antibodies to PD-1, PDL1 and PDL2 are known in the art. For example, the anti-PD-1 antibody used in the present invention may be selected from those described in Ohaegbulam et al. (Ohaegbulam et al, 2015), all contents of which are incorporated herein by reference. Examples of human or humanized anti-PD-1 antibodies include, but are not limited to, CD279 (human monoclonal anti-PD1 antibody, Bio X Cell), MEDI0680 (AMP-514, anti-PD-1 humanized monoclonal anti-IgG4 antibody, AstraZeneca), Nivolumab (BMS-936558, anti-PD1 human IgG4 antibody, Bristol-Myers Squibb), Pembrolizumab (Lambrolizumab, MK-3475, anti-PDI humanized anti-IgG4 monoclonal antibody, Merck), Pidilizumab (ST-011, anti-PDI humanized anti-IgG1 monoclonal antibody, Medivation) and TSR-042 (anti-PD-1 humanized anti-IgG4 monoclonal antibody, Tesaro). Examples of human or humanized anti-PDL1 antibodies include, but are not limited to, Avelumab (MSB0010718C, anti-PDL1 anti-human IgG1 monoclonal antibody, Merck-Serono), Atezolizumab (MPDL3280A, RG7446, Hoffmann-La Roche anti-PDL1 anti-human IgG monoclonal antibody) , BMS-936559 (MDX-1105, anti-PDL1 humanized monoclonal antibody to human IgG4, Bristol-Myers Squibb), Durvalumab (MEDI4736, anti-PDL1 humanized monoclonal antibody to IgG1, AstraZeneca), KN035 (anti-PDL1 monoclonal antibody, 3D Medicines ) and LY3300054 (anti-PDL1 monoclonal antibody, Eli Lilly). Examples of human or humanized anti-PDL2 antibodies include, but are not limited to, AMP-224 (IgG2a PDL2 Fc fusion protein, AstraZeneca). In some embodiments, an anti-PD-1 antibody, an anti-PDL1 antibody, and/or an anti-PDL2 antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0. 3 mg/kg - 15 mg/kg, 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1, 5 mg/kg - 10 mg/kg, 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[56] In some embodiments, a human may be administered Pidilizumab at a dosage of 0.2-6 mg/kg or 1.5-6 mg/kg; Pembrolizumab can be administered to humans at a dosage of 1-10 mg/kg; Nivolumab can be administered to humans at a dosage of 0.3-20 mg/kg, 0.3-10 mg/kg, 1-10 mg/kg, or 1 or 3 mg/kg; BMS-936559 can be administered to humans at a dosage of 0.3-10 mg/kg; Atezolizumab can be administered to humans at a dosage of 1-20 mg/kg; Durvalumab can be administered to humans at a dosage of 0.1-15 mg/kg; and Avelumab can be administered to humans at a dosage of 1-20 mg/kg.

[57] T cell immunoglobulin and mucin domain-3 (TIM-3) is a Th1-specific cell surface protein that acts as an immune checkpoint that inhibits lymphocyte activity by down-regulating macrophage activation and playing important role in the exhaustion of CD8+ T cells that occurs in chronic pathological conditions of the immune system. TIM-3 acts as a negative regulator of Th1/TC1 function by inducing cell death upon interaction with its ligand, galectin-9 (Gal9). Essentially, compounds that inhibit TIM-3 function, such as TIM-3 inhibitors and/or PD-Gal9 inhibitors, serve to activate the immune system. One class of TIM-3 inhibitors includes antagonistic or neutralizing antibodies to TIM-3 and/or Gal-9. Many antagonistic or neutralizing antibodies to TIM-3 and Gal-9 are known in the art. Examples of human or humanized antibodies to TIM-3 include, but are not limited to, AF2365 (anti-TIM-3 monoclonal antibody to human IgG, R&D Systems), CD366 (anti-TIM-3 monoclonal antibody to human IgG1, BioLegend), F38-2E2 (anti-TIM-3 monoclonal antibody to IgG1, R&D Systems), L3D (anti-TIM-3 monoclonal antibody to human IgG1, CN 102492038 B), MAB2365 (anti-TIM-3 monoclonal antibody to IgG2a, R&D Systems), MAB23651 (anti-TIM-3 anti-human IgG1 monoclonal antibody, R&D Systems) and TSR-022 (humanized anti-TIM-3 anti-IgG4 monoclonal antibody, Tesaro). In some embodiments, the anti-TIM-3 antibody and/or anti-Gal9 antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0. 3 mg/kg - 15 mg/kg, 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1, 5 mg/kg - 10 mg/kg, 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[58] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as cluster of differentiation 152 (CD152), is a protein receptor that functions as an immune checkpoint that negatively regulates (down-regulates) the immune response. CTLA-4 is constitutively expressed in Treg cells but is up-regulated only in conventional T cells upon activation. CTLA-4 acts as an "on switch" when bound to CD80 or CD86 on the surface of antigen presenting cells. Essentially, compounds that inhibit CTLA-4 function, such as CTLA-4 inhibitors, CD80 inhibitors and/or CD86 inhibitors, serve to activate the immune system. One class of CTLA-4 inhibitors includes antagonistic or neutralizing antibodies to CTLA-4, CD80 and/or CD86. Many antagonistic or neutralizing antibodies to CTLA-4, CD80 and CD86 are known in the art. Examples of human or humanized anti-CTLA-4 antibodies include, but are not limited to, Ipilimumab (anti-CTLA-4 human IgG1 monoclonal antibody, Bristol-Myers Squibb) and Tremelimumab (anti-CTLA-4 anti-human IgG2 monoclonal antibody, Pfizer). In some embodiments, the anti-CTLA-4 antibody, anti-PD-CB80 antibody, and/or anti-PD-CD86 antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/ kg, 0.3 mg/kg - 15 mg/kg, 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg /kg - 15 mg/kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/ kg, 1.5 mg/kg - 10 mg/kg, 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[59] Killer cell immunoglobulin-like receptors (KIRs) are a family of type I transmembrane glycoproteins expressed on the plasma membrane of natural killer cells (NK cells) and a minority of T cells. They regulate the killing function of these cells by interacting with class I major histocompatibility complex (MHC) molecules, which are expressed on all types of nucleated cells. Thus, KIRs are inhibitors of lymphocyte activity. Essentially, compounds that inhibit KIR function, such as KIR inhibitors, serve to activate the immune system. One class of KIR inhibitors includes antagonistic or neutralizing antibodies to KIR. Many antagonistic or neutralizing antibodies to KIR are known in the art. Examples of human or humanized anti-KIR antibodies include, but are not limited to, Lirilumab (BMS-986015, human anti-KIR monoclonal antibody, Bristol-Myers Squibb). In some embodiments, the anti-KIR antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg /kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg/kg - 15 mg/kg , 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg/kg - 10 mg/kg , 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[60] Lymphocyte activating gene 3 (LAG-3), also known as cluster of differentiation 223 (CD223), is a cell surface molecule with various biological effects on T cell function. LAG-3 is an immune checkpoint that inhibits lymphocyte activity by suppressing the immune response by targeting Treg cells as well as directly affecting CD8+ T cells. Essentially, compounds that inhibit LAG-3 function, such as LAG-3 inhibitors, serve to activate the immune system. One class of LAG-3 inhibitors includes antagonistic or neutralizing antibodies to LAG-3. Many antagonistic or neutralizing antibodies to LAG-3 are known in the art. Examples of human or humanized anti-LAG-3 antibodies include, but are not limited to, BMS-986016 (human anti-LAG-3 monoclonal antibody, Bristol-Myers Squibb). In some embodiments, the anti-LAG-3 antibody may be administered to a human at a dosage of, for example, 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg /kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg/kg - 15 mg/kg , 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg/kg - 10 mg/kg , 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[61] OX40, also known as cluster of differentiation 134 (CD134), is a member of the TNFR receptor superfamily. OX40 promotes the expansion of effector and memory T cells, but is also known to suppress the differentiation and activity of regulatory T cells, as well as the regulation of cytokine production. Being transiently expressed after T-cell receptor engagement, OX40 is up-regulated only on those T cells that are later to be activated by antigen in inflammatory lesions. Its ligand is OX40L, also known as cluster of differentiation 252 (CD252). Essentially, compounds that activate or stimulate OX40 function, such as OX40 activators and/or OX40L activators, serve to activate the immune system. One class of OX40 activators includes agonist antibodies to OX40 and OX40L. There are many agonist antibodies to OX40 and OX40L known in the art. Examples of human or humanized anti-OX40 antibodies include, but are not limited to, GSK3174998; humanized anti-OX40 monoclonal antibody to IgG1; GlaxoSmithKline), MEDI0562 (humanized anti-OX40 monoclonal antibody, MedImmune) and MEDI6383 (human OX40 fusion protein, MedImmune). Other anti-OX40 antibodies include, but are not limited to, MEDI6469 (9 B12, mouse OX40 monoclonal antibody, MedImmune). In some embodiments, the anti-OX40 antibody and/or anti-OX40L antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0. 2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg /kg - 15 mg/kg, 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/ kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg /kg - 10 mg/kg, 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[62] Anti-GITR antibodies target the glucocorticoid-induced tumor necrosis factor receptor (GITR) protein, which is regularly expressed on the surface of regulatory T cells (Treg cells) and on the surface of effector T cells after their activation. Anti-GITR antibodies block the interaction of GITR, found on various types of T cells, with its ligand, thereby inducing both the activation of tumor-antigen-specific T effector cells and the reversal of suppression induced by inappropriately activated T regulatory cells. Essentially, compounds that activate or stimulate GITR function, such as GITR activators, serve to activate the immune system. One class of GITR activators includes agonist antibodies to GITR. Many agonist antibodies to GITR are known in the art. Examples of human or humanized anti-TIGR antibodies include, but are not limited to, GWN323 (humanized anti-GITR monoclonal antibody; Novartis) and TRX518 (humanized anti-GITR monoclonal antibody; GITR, Inc.). In some embodiments, the anti-GITR antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg /kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0.2 mg/kg - 10 mg/kg , 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg/kg - 15 mg/kg, 0 .3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/kg, 1 mg/kg - 10 mg /kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg/kg - 10 mg/kg, 1 .5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[63] CD27 is a member of the tumor necrosis factor receptor superfamily. CD27 activity is regulated by the transient availability of its ligand, CD70, on lymphocytes and dendritic cells. CD27 activation plays a key role in regulating B cell activation and immunoglobulin synthesis, supports antigen-specific expansion of naïve T cells, is essential for the generation and long-term maintenance of T cell immunity, and is a marker of B cell memory. CD27 transmits signals leading to the activation of NF-κB and MAPK8/JNK. As such, compounds that activate or promote CD27 function, such as CD27 activators and/or CD70 activators, serve to activate the immune system. One class of CD27 activators includes agonist antibodies to CD27 and/or CD70. There are many agonist antibodies to CD27 and/or CD70 known in the art. Examples of human or humanized anti-CD27 antibodies include, but are not limited to, Varlilumab (CDX-1127, human anti-CD27 monoclonal antibody, Celldex Therapeutics). In some embodiments, the anti-CD27 antibody and/or anti-CD70 antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0. 2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg /kg - 15 mg/kg, 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/ kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg /kg - 10 mg/kg, 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[64] Inducible T cell costimulator (ICOS), also known as cluster of differentiation 278 (CD278), is a cell surface costimulatory receptor of the CD28 superfamily that is expressed on activated T cells. It is an activator of T cell function. Essentially, compounds that activate or stimulate ICOS function, such as ICOS activators and/or B7RP1 activators, serve to activate the immune system. One class of ICOS activators includes agonist antibodies to ICOS and/or B7RP1. There are many agonist antibodies to ICOS and B7RP1 known in the art. In some embodiments, the anti-ICOS antibody and/or anti-B7RP1 antibody may be administered to a human at a dosage of, for example, about 0.1 mg/kg - 20 mg/kg, 0.1 mg/kg - 15 mg/kg, 0.1 mg/kg - 10 mg/kg, 0.1 mg/kg - 5 mg/kg, 0.2 mg/kg - 20 mg/kg, 0.2 mg/kg - 15 mg/kg, 0. 2 mg/kg - 10 mg/kg, 0.2 mg/kg - 6 mg/kg, 0.2 mg/kg - 5 mg/kg, 0.3 mg/kg - 20 mg/kg, 0.3 mg /kg - 15 mg/kg, 0.3 mg/kg - 10 mg/kg, 0.3 mg/kg - 5 mg/kg, 1 mg/kg - 20 mg/kg, 1 mg/kg - 15 mg/ kg, 1 mg/kg - 10 mg/kg, 1 mg/kg - 5 mg/kg, 1.5 mg/kg - 20 mg/kg, 1.5 mg/kg - 15 mg/kg, 1.5 mg /kg - 10 mg/kg, 1.5 mg/kg - 6 mg/kg or 1.5 mg/kg - 5 mg/kg.

[65] In some embodiments, combinations of antibodies may be used, among others: CT-011 in combination with Rituximab (trade names Rituxan, MabThera and Zytux), a chimeric monoclonal antibody to the CD20 protein, for example, 3 mg/day. kg each; Nivolumab (eg, 1 mg/kg) in combination with ipilimumab; for example, 3 mg/kg); or Nivolumab (eg, 1–10 mg/kg) in combination with the limited multipeptide vaccine HLA-A*0201 (Weber et al, 2013).

[66] In some embodiments, the active agent that can be used in the present invention can be a mimic antibody. Antibody mimics can specifically bind antigens like antibodies, but they are not structurally related to antibodies. They are typically artificial peptides or proteins with a molar mass of about 3 to 20 kDa. In aspects of the present embodiment, the disclosed antibody mimetic may be an affibody molecule; affiliate; affimer; affitin; alphabody; anticalin; avimer; Darpin; finomere; Kunitz domain peptide; or monobody. Non-limiting examples of mimetic antibodies are presented in Table 1:

[67] In some embodiments, the active agent that can be used in the present invention may be an aptamer. Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule. In aspects of the present embodiment, the aptamer described herein may be a DNA aptamer, an RNA aptamer, an XNA aptamer, or a peptide aptamer.

[68] In some embodiments, an antibody described herein may be in combination with an adjuvant. Adjuvant refers to any substance or mixture of substances that enhances or diversifies the immune response. An adjuvant may serve to reduce the number of immunizations or the amount of antigen required for protective immunization. Adjuvants include, but are not limited to, such as, for example, liposomes, oil phases, including, without limitation, Freund's type adjuvants, such as, for example, Freund's complete adjuvant (FCA); Freund's incomplete adjuvant (FIA); sapogenin glycosides, such as, for example, saponins; carbopol; N-acetylmuramyl-L-alanyl-D-isoglutamine (commonly known as muramyl dipeptide or "MDP"); and lipopolysaccharide (LPS). Such adjuvants are usually used in the form of an emulsion with an aqueous phase, or, more often, with water-insoluble inorganic salts. These inorganic salts include aluminum hydroxide, zinc sulfate, colloidal iron hydroxide, calcium phosphate or calcium chloride. In aspects of this embodiment, the antibody described herein can be combined, for example, with an anti-CTLA-4 antibody in combination with an anti-OX40 antibody and a TLR9 ligand such as CpG (Marabelle et al, 2013).

[69] In some embodiments, the active agent that can be used in the present invention can be a small molecule. In aspects of this embodiment, the small molecule disclosed herein may be: (a) a p300 inhibitor (Liu et al, 2013), such as gemcitabine (low dose), C646, or analogs thereof, i.e., a compound of Formula I:

wherein selected from H, -CO ₂ R ₆ , -CONR ₆ R ₇ , -SO ₃ H or -SO ₂ NR ₆ R ₇ ; R ₂ is selected from H, -CO ₂ R ₆ or halogen, preferably Cl; R ₃ is selected from halogen, preferably F, -NO ₂ , -CN, -CO ₂ R ₆ , preferably CO ₂ CH ₃ or CO ₂ CH ₂ CH ₃ or -CH ₂ OH; R ₄ and R ₅ are each independently H or -C ₁ -C ₆ alkyl, preferably methyl; R ₆ represents H or -C ₁ -C ₆ alkyl, preferably H, methyl or ethyl; and R ₇ represents H or -C ₁ -C ₆ alkyl, preferably H or methyl; (b) Sunitinib; (c) Polyoxomthalate-1 (POM-1) (Ghiringhelli et al, 2012); (d) α,β-methylenadenosine 5'-diphosphate (APCP); (e) arsenic trioxide (As ₂ O ₃ ); (f) GX15-070 (Obatoclax); (g) a retinoic acid antagonist such as Ro 41-5253 (a synthetic retinoid and selective small molecule antagonist) or LE-135; (h) a SIRPα (CD47) antagonist such as CV1-hIgG4 (SIRPα variant) alone or in combination with an anti-CP47 antibody; (i) a CCR4 antagonist, such as AF399/420/18025, alone or in combination with an anti-CCR4 antibody; (j) an adenosine receptor antagonist; (k) adenosine A1 receptor antagonist; adenosine receptor A2a; (m) adenosine A2b receptor antagonist; (n) adenosine A3 receptor antagonist; (o) indoleamine 2,3-dioxygenase antagonist; or (p) HIF-1 regulator.

[70] In some embodiments, the agent is a p300 inhibitor, the formulas of which are shown in Table 2, i.e. C646 (4-(4-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazole-1 -yl)benzoic acid), C146 (4-hydroxy-3-(((2-(3-iodophenyl)benzo[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, C646 is an inhibitor of p300.

[71] In some embodiments, the adenosine receptor antagonist may be CGS15943 (9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazoline-5-amine); the antagonist of the adenosine A1 receptor can be PSB 36 (1-Butyl-8-(hexahydro-2,5-methanopantenan-3a(1H)-yl)-3,7-dihydro-3-(3-hydroxypropyl)-1H-purine- 2,6-dione); an antagonist of the adenosine A2a receptor may be SCH58261 (5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c )pyrimidine), SYN115 (4-hydroxy-N-[4-methoxy-7-(4-morpholinyl)-2-benzothiazolyl]-4-methyl-1-piperidinecarboxamide), FSPTP (also called SCH58261 (5-amino-7 -[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine), SCH442416 (2- (2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine) , or ZM241385 (also called tosadentant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidin-1-carboxamide), an adenosine A2b receptor antagonist may be PSB 603 (8 {4-[4-(4-chlorophenyl)piperazine-1-sulfonyl]phenyl}-1-propyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione (Nakatsukasa et al, 2011)), and the A3 receptor antagonist may be MRS3777 (2-phenoxy-6-(cyclohexylamino)purine hemioxalate).

[72] In some embodiments, the small molecule inhibitor of the indoleamine 2,3-dioxygenase pathway may be indoximod (NSC-721782/NLG-9189 (1-methyl-D-tryptophan), NewLink Genetics), INCB024360 ((4E)-4- [(3-chloro-4-fluoroanilino)nitrosomethylidene]-1,2,5-oxadiazol-3-amine, Incyte) or NLG-919 (1-Cyclohexyl-2-(5H-imidazo[5,1-a]isoindole -5-yl)ethanol), NewLink Genetics).

[73] The HIF-1 regulator may be M30, (5-N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), described in Zheng et al. (Zheng et al, 2015).

[74] In some embodiments, an active agent that can be used in the present invention can be any combination of an antibody disclosed herein and a small molecule disclosed herein. In aspects of this embodiment, the active agent can be any combination of an antibody described herein and a small molecule described herein.

[75] In some embodiments, the active agent to be used in the present invention may be a protein selected from the group consisting of: (a) neem leaf glycoprotein (NLGP; (Roy et al, 2013)); and/or (b) sCTLA-4 (soluble isoform of CTLA-4) (Ward et al, 2013).

[76] In some embodiments, the active agent that can be used in the present invention can be a silencing molecule. In aspects of this embodiment, the silencing molecule is selected from the group consisting of antisense miR-126 (Qin et al, 2013) and anti-galectin-1 (Gal-1; (Dalotto-Moreno et al, 2013)).

[77] In some embodiments, the active agent that can be used in the present invention may be OK-432 (lyophilized preparation of Streptococcus pyogenes) (Hirayama et al, 2013).

[78] In some embodiments, the active agent that can be used in the present invention can be a combination of IL-12 and anti-CTLA-4.

[79] In some embodiments, the agent may be a broad spectrum antibiotic derivative that targets gram-positive and gram-negative bacteria, thereby promoting immunomodulation of regulatory T cells, for example, vancomycin, which targets gram-positive bacteria and has been shown to reduce Treg cell level/activity.

[80] In some embodiments, the active agent that can be used in the present invention can be any combination of an antibody disclosed herein, an antibody mimetic disclosed herein, an aptamer disclosed herein, a small molecule disclosed herein, neem leaf glycoprotein disclosed herein, sCTLA-disclosed herein. 4, the silencing molecule disclosed herein, OK-432 disclosed herein, and/or the combination of IL-12 and anti-CTLA-4 disclosed herein.

[81] As discussed above, the active agent is administered in a dosage regimen containing at least two courses of treatment, with each course of treatment comprising a treatment session followed by a rest period.

[82] The terms “treatment session” and “treatment period” are used interchangeably herein and refer to the period during which one or more of the active agents described herein is administered to the patient. As will be discussed in more detail below, a treatment session may be a single dosing regimen or a multiple dosing regimen administered over a period of time. The treatment session is such that a therapeutically effective amount of the active agent described herein is maintained throughout.

[83] The terms “non-treatment period”, “treatment break”, “non-treatment period”, “break” are used interchangeably herein and refer to the period during which the active agent described herein is not administered to a patient undergoing treatment. Cessation of administration of the active agent during the non-treatment period results in the reduction of the active agent described herein to subtherapeutic levels in the patient undergoing treatment. As described herein, a “non-treatment period” is not the same event as the period of time between dosages constituting a multiple dosing regimen that occurs during a treatment session. If the administration of one or more active agents described herein during a treatment session is repeated, the non-treatment period will be longer than the intervening period between these repeated administrations during the treatment session.

[84] The dosage regimen can be determined in several ways. For example, the level of immunosuppression can be calibrated to the desired level for each patient being treated (personalized medicine) by monitoring the level or activity of IFN-γ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation individually, and adjusting the treatment session, frequency of administration and break empirically and individually, as determined by monitoring results.

[85] Thus, a treatment session may comprise administering an active agent or pharmaceutical composition to a patient, and the treatment session continues at least until the systemic presence or level of IFN-γ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation rises above the reference value, then administration is suspended for the duration of the break, and the break continues as long as that level is above the reference value, the reference value being selected from (a) the level of systemic presence or activity of IFN-γ-producing leukocytes or the rate of leukocyte proliferation in response to stimulation measured in the last blood sample obtained from the patient before said administration; or (b) the level of systemic presence or activity of IFN-γγ-producing leukocytes, or the rate of leukocyte proliferation in response to stimulation, characteristic of a group of individuals suffering from a disease, disorder, pathological condition or lesion of the central nervous system.

[86] The duration of treatment sessions and non-treatment sessions or breaks can be determined by clinicians in clinical trials targeting specific groups of patients and then applied sequentially to that group of patients, without the need for individual monitoring of the level of immunosuppression.

[87] In some embodiments, a treatment session comprises administering an active agent to a patient, and continues at least until the systemic presence of the active agent reaches a therapeutic level, the administration is suspended during the break, and the break continues until the level remains above 95%, 90%, 80%, 70%, 60% or 50% of said therapeutic level. The term “therapeutic level” is used herein to refer to the conventional systemic level of drugs used to block immune checkpoints in known treatments such as cancer therapy (see above).

[88] In some embodiments, a treatment session comprises administering an active agent to a patient, and continues at least until the systemic presence or activity of the active agent reaches a therapeutic level, at which point administration is suspended and the interruption continues until systemic the presence or activity of the active agent remains above the therapeutic threshold. In aspects of this embodiment, the therapeutic threshold level is a level of, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, not less than 80%, not less than 90% of the therapeutic level. The term "therapeutic level" is used herein to refer to the conventional systemic level of drugs used to block immune checkpoints in established therapies such as cancer therapy (see above). In aspects of this embodiment, the active agent is an anti-PD-1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-CTLA-4 antibody, anti-B7RP1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-B7-H7 antibody, anti-BTLA antibody, anti-HVEM antibody, anti-CD-27 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD80 antibody, anti-CD86 antibody , anti-CD137 antibody, anti-CD137L antibody, anti-OX40 antibody, anti-OX40L antibody, anti-TIM-3 antibody, anti-Galectin 9 antibody, anti-KIR antibody, anti-LAG-3 antibody, anti-ICOS antibody , anti-VISTA antibody, anti-STING, anti-TIGIT, anti-GITR, or any combination thereof.

[89] In some embodiments, a treatment session comprises administering an active agent to a patient, and continues at least until the systemic presence or activity of the active agent reaches a therapeutic level, at which point administration is suspended and the interruption continues until positive the effect on cognitive performance remains above pre-treatment levels. In aspects of this embodiment, a positive effect on cognitive performance is maintained that shows an improvement of, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% above pre-treatment cognitive level. In aspects of this embodiment, the active agent is an anti-PD-1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-CTLA-4 antibody, anti-B7RP1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-B7-H7 antibody, anti-BTLA antibody, anti-HVEM antibody, anti-CD-27 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD80 antibody, anti-CD86 antibody , anti-CD137 antibody, anti-CD137L antibody, anti-OX40 antibody, anti-OX40L antibody, anti-TIM-3 antibody, anti-Galectin 9 antibody, anti-KIR antibody, anti-LAG-3 antibody, anti-ICOS antibody , anti-VISTA antibody, anti-STING, anti-TIGIT, anti-GITR, or any combination thereof.

[90] In some embodiments, a treatment session comprises administering an active agent to a patient, and continues at least until the systemic presence or activity of the active agent reaches a therapeutic level, at which point administration is suspended and the interruption continues until positive the effect on vision remains above pre-treatment levels. In aspects of this embodiment, a beneficial effect on vision is maintained that shows an improvement of, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90% higher than the level of vision before treatment. In aspects of this embodiment, the active agent is an anti-PD-1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-CTLA-4 antibody, anti-B7RP1 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-B7-H7 antibody, anti-BTLA antibody, anti-HVEM antibody, anti-CD-27 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD80 antibody, anti-CD86 antibody , anti-CD137 antibody, anti-CD137L antibody, anti-OX40 antibody, anti-OX40L antibody, anti-TIM-3 antibody, anti-Galectin 9 antibody, anti-KIR antibody, anti-LAG-3 antibody, anti-ICOS antibody , anti-VISTA antibody, anti-STING, anti-TIGIT, anti-GITR, or any combination thereof.

[91] In some embodiments, a treatment session may be a single administration or comprise multiple administrations performed over a predetermined period of time. In aspects of this embodiment, a treatment session may be multiple administrations over, for example, 1 day to four weeks, 2 days to four weeks, 3 days to four weeks, 4 days to four weeks, 5 days to four weeks, 6 days to four weeks, one week and four weeks, 10 days and four weeks, two weeks and four weeks, 17 days and four weeks, or three weeks and four weeks. For example, a treatment session may contain two administrations, both within one week, for example if the second administration occurs on the 1st, 2nd, 3rd, 4th, 5th or 6th day after the first. As another example, a treatment session may comprise three administrations, all within one week, for example, 1, 2 or 3 days after the previous administration. As another example, a treatment session may comprise three administrations, all within two weeks, for example, 1, 2, 3, 4 or 5 days after the previous administration. As another example, a treatment session may comprise four administrations, all within two weeks, for example, 1, 2, 3, 4 or 5 days after the previous administration. As another example, a treatment session may comprise four administrations, all within three weeks, for example, 1, 2, 3, 4, 5 or 6 days after the previous administration. As another example, a treatment session may comprise five administrations, all within three weeks, for example, 1, 2, 3, 4 or 5 days after the previous administration.

[92] In some embodiments, the treatment-free interval may range from one week to six months, e.g., 2 weeks to 4 weeks, 3 weeks to 4 weeks, 2 weeks to 6 weeks, 3 weeks to 6 weeks, 4 weeks to 6 weeks, from 5 weeks to 6 weeks, from 2 weeks to 2 months, from 3 weeks to 2 months, from 4 weeks to 2 months, from 5 weeks to 2 months, from 6 weeks to 2 months, from 7 weeks up to 2 months, from 2 months to 3 months, from 2 months to 4 months, from 3 months to 4 months, from 3 months to 5 months, from 3 months to 5 months, from 4 months to 5 months, from 1 week to 6 months, from 2 weeks to 6 months, from 3 weeks to 6 months, 4 weeks to 6 months, from 6 weeks to 6 months, from 2 months to 6 months, from 3 months to 6 months, from 4 months to 6 months , or from 5 months to 6 months. In some embodiments, the non-treatment interval may be 1 to 2 months, 1 to 3 months, or 2 to 3 months.

[93] During a treatment session, administration of the active agent or pharmaceutical composition may be a single administration or repeated administration, for example, the active agent or pharmaceutical composition may be administered only once followed immediately by a rest period, or it may be administered daily or once at a time every two, three, four, five or six days, once a week, every two weeks, every three weeks or every four weeks. This frequency is applicable to any active agent, may be based on generally accepted practices in the art, and may be ultimately determined by physicians in clinical trials. In addition, the frequency of repeated administrations during a treatment session may vary depending on the nature of the active agent, with, for example, small molecules being administered daily and antibodies being administered every 3 days. It should be understood that when the agent is administered during a treatment session at a relatively low frequency, for example, once a week for a one-month treatment session, or once a month for a six-month treatment session, this treatment session is followed by a rest period, the duration of which is greater than the interval between repeated administrations during a treatment session (ie in this example it is, respectively, more than one week or one month). A period of one week or one month between administrations during a treatment session is not considered a break in treatment in this example.

[94] The duration of the treatment session and the non-treatment session or break may be adjusted according to the frequency of administration, so that, for example, the frequency of administration of the active agent once every 3 days may result in a treatment session duration of 6 or 9 days, and a corresponding break.

[95] If the treatment session consists of a single administration, the dosage regimen is determined by the length of the no-treatment interval, so that one administration is followed by a no-treatment interval of 7, 8, 9, 10, 14, 18, 21, 24 or 28 days or longer before the next treatment session with a single injection. Specifically, the dosage regimen consists of separate administrations alternating with no-treatment intervals of 2, 3 or 4 weeks. In addition, the dosage regimen may consist of separate administrations alternating with no treatment intervals of 2 to 4 weeks, 2 to 3 weeks, or 3 to 4 weeks.

[96] If a treatment session consists of several administrations, the dosage regimen is determined by the duration of the non-treatment interval, so that several administrations within one week are followed by a non-treatment interval of 7, 10, 14, 18, 21, 24 or 28 days or longer before the next treatment session with several injections. Specifically, the dosage regimen may consist of multiple administrations over the course of one week, interspersed with no-treatment intervals of 2, 3, or 4 weeks. In addition, the dosage regimen may consist of multiple administrations over one week, alternating with no treatment intervals of 2 to 4 weeks, 2 to 3 weeks, or 3 to 4 weeks.

[97] As another example, a dosage regimen may comprise multiple administrations over two weeks, followed by a no-treatment interval of 2 weeks, 3 weeks, or 1, 2, 3, or 4 months or longer, until the next multiple administration treatment. Specifically, the dosage regimen may consist of multiple administrations over two weeks, interspersed with no-treatment intervals of 1, 2, 3, or 4 months. In addition, the dosing regimen may consist of multiple administrations over two weeks with no treatment intervals of 1 to 2 months, 1 to 3 months, 1 to 4 months, 2 to 3 months, 2 to 4 months, or 3 up to 4 months.

[98] As another example, a dosage regimen may comprise multiple administrations over three weeks, followed by a treatment-free interval of 1, 2, 3, 4, 5 or 6 months or longer, until the next multiple administration treatment. Specifically, the dosage regimen may consist of multiple administrations over three weeks, interspersed with no-treatment intervals of 1, 2, 3, 4, 5, or 6 months. In addition, the dosage regimen may consist of multiple administrations over three weeks with no treatment intervals of 1 to 2 months, 1 to 3 months, 1 to 4 months, 1 to 5 months, 1 to 6 months, 2 up to 3 months, from 2 to 4 months, from 2 to 5 months, from 2 to 6 months, from 3 to 4 months, from 3 to 5 months, from 3 to 6 months, from 4 to 5 months, from 4 to 6 months, or from 5 to 6 months.

[99] Of course, there is a flexible dosing regimen that starts with a certain regimen and changes to another. For example, treatment sessions each comprising 2 separate administrations 3 days apart, with an interval of, for example, 1 week between treatments, may be replaced, if deemed appropriate, by a dosing regimen comprising treatment sessions with single administrations , separated by breaks lasting, for example, 2, 3 or 4 weeks. As another example, treatment sessions each comprising 2 separate administrations 7 days apart, with an interval of, for example, 2 weeks between treatment sessions, may be replaced, if deemed appropriate, by a dosage regimen comprising treatment sessions with single administrations separated by intervals of, for example, 2, 3, 4, 5 or 6 weeks. As another example, treatment sessions each comprising 3 separate administrations 3 days apart, with an interval of, for example, 2 weeks between treatments, may be replaced, if deemed appropriate, by a dosage regimen comprising treatment sessions with single administrations separated by intervals of, for example, 2, 3, 4, 5 or 6 weeks.

[100] In any case, the dosage regimen, that is, the duration of the treatment session and the break, is selected so as to reduce the level of immunosuppression, for example, as measured by a decrease in the level of systemic presence or activity of regulatory T cells, or an increase in the patient's level of systemic presence or activity leukocytes producing IFN-γ was short-lived.

[101] The method, active agent or pharmaceutical composition in accordance with the present invention can be used for treating a disease, disorder or condition of the CNS that is a neurodegenerative disease, disorder or condition. According to aspects of the present embodiment, neurodegenerative diseases, disorders or conditions include Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis; secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, tauopathy, retinal degeneration; anterior ischemic optic neuropathy; glaucoma; uveitis; depression; Trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, diffuse Lewy body disease, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia, or progressive supranuclear palsy. In some embodiments, the CNS condition is age-related dementia. In some aspects of this embodiment, CNS pathological conditions include Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease.

[102] Tauopathies are a clinically, morphologically, and biochemically heterogeneous class of neurodegenerative diseases characterized by the pathological aggregation of tau protein into neurofibrillary or gliofibrillary tangles in the human brain. Tau, a microtubule-associated protein (MAP), binds to microtubules and promotes their polymerization. It plays an important role in maintaining axonal trafficking and neuronal integrity, but has a physiological role in dendrites and is expressed at low levels in glial cells. In tauopathy, bundles are formed by hyperphosphorylation of tau, causing it to aggregate in an insoluble form. Examples of tauopathies include, but are not limited to, Alzheimer's disease, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, boxer's dementia, frontotemporal dementia, frontotemporal degeneration, Hallerwarden-Spatz disease, Huntington's disease, ganglioglioma, gangliocytoma, globular glial tauopathy , lead encephalopathy, lipofuscinosis, Lytic-Bodiga disease (Parkinson's disease and dementia complex in Guam), meningoangiomatosis, parkinsonism associated with chromosome 17, Pick's disease, primary age-related tauopathy (PART), formerly known as dementia with neurofibrillary tangles (NFT- dementia), postencephalitic parkinsonism, progressive supranuclear palsy, subacute sclerosing panencephalitis and tuberous sclerosis.

[103] Retinal degeneration disorders result in retinal degradation due to the death of photoreceptor cells. There are several causes of retinal degeneration, including artery or vein occlusion, diabetic retinopathy, retrolental fibroplasia/retinopathy of prematurity, or disease (usually hereditary). Symptoms include, but are not limited to, blurred vision, night blindness, retinal detachment, light sensitivity, glare sensitivity, tunnel vision, loss of depth perception, loss of contrast, night blindness, loss of central vision, loss of peripheral vision, and complete loss of vision. Retinal degeneration disorders include, but are not limited to, age-related macular degeneration (wet and dry), retinitis pigmentosa, choroideremia, cone-rod retinal degeneration, gyrate atrophy, juvenile retinoschisis, icteric macular degeneration (Best's disease), abetalipoproteinemia (Bassen's disease). Kornzweig), Bardet-Biedl syndrome, blue cone monochrome syndrome, dominant drusen, Goldmann-Favre vitreoretinal dystrophy (enhanced S-cone syndrome), Kearns-Sayre syndrome, Lawrence-Moon syndrome, Leber congenital amaurosis, Refsum Leber disease, Oguchi disease, peripapillary (pericentral) choroidal dystrophy, pigmentary dystrophy, Sorsby macular dystrophy, Stargardt disease, Stickler syndrome, Usher syndrome and Wagner vitreoretinal dystrophy.

[104] In some embodiments, each of the above-described active agents blocking one of the immune checkpoints, including ICOS-B7RP1, V-domain immunoglobulin suppressor of T-cell activation (VISTA), B7-C028-like molecule, CD40L- CD40, CD28-CD80, CD28-CD86, B7H3, B7H4, B7H7, BTLA-HVEM, CD137-CD137L, OX40L, CD27-CD70, STING, TIGIT and A2aR-adenosine, as well as indoleamine 2,3-dioxygenase (IDO) -L-tryptophan, such as an antibody to one of two immune checkpoint components, is indicated for the treatment of neurodegenerative diseases, disorders or conditions including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis; secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, retinal degeneration selected from the group that includes age-related macular degeneration and retinal pigmentary degeneration; anterior ischemic optic neuropathy; glaucoma; uveitis; depression; Trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, diffuse Lewy body disease, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia, or progressive supranuclear palsy. Treatment of any of these diseases using any of these active agents can be carried out according to the regimen described above.

[105] The method, active agent and pharmaceutical composition in accordance with the present invention can be further used in the treatment of injuries of the central nervous system, including spinal cord injury, closed head injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebral ischemia, trauma optic nerve, myocardial infarction, organophosphate poisoning and trauma caused by tumor removal.

[106] In some embodiments, each of the active agents described above blocks one of the immune checkpoints, including ICOS-B7RP1, V-domain immunoglobulin suppressor of T-cell activation (VISTA), B7-CD28-like molecule, CD40L- CD40, CD28-CD80, CD28-CD86, B7H3, B7H4, B7H7, BTLA-HVEM, CD137-CD137L, OX40L, CD27-CD70, STING, TIGIT and A2aR-adenosine, as well as indoleamine 2,3-dioxygenase (IDO) -L-tryptophan, such as an antibody to one of the two immune checkpoint components, is indicated for the treatment of CNS injuries, including spinal cord injury, closed head injury, blunt trauma, penetrating trauma, hemorrhagic stroke, ischemic stroke, cerebral ischemia, injury optic nerve, myocardial infarction, organophosphate poisoning and trauma caused by tumor removal. Treatment of any of these injuries using any of these active agents can be carried out according to the regimen described above.

[107] As stated above, the inventors have found that the present invention improves cognitive function in Alzheimer's disease-mimicking mice. Thus, this method, active agent and pharmaceutical composition can be used to improve motor and/or cognitive function of the central nervous system, for example, to reduce age-related loss of cognitive function that can occur in people without diagnosed diseases, as well as in people suffering from neurodegenerative diseases. In addition, the method, active agent and pharmaceutical composition can be used to reduce the loss of cognitive function resulting from acute stress or traumatic event. The cognitive functions mentioned above may include learning, memory, or both.

[108] It should be emphasized that improvement of cognitive functions in mice emulated with Alzheimer's disease (5XFAD AD-Tg mice) was observed and described by the inventors at various stages of disease manifestation; In both early and late stages of the disease, pathology can be reduced through treatment. 5XFAD AD-Tg mice begin to exhibit cerebral plaque pathology at 2.5 months of age and cognitive impairment at 5 months of age (Oakley et al, 2006). It should be noted that while in Example 2 below the inventors describe a therapeutic effect in 5XFAD mice at 6 months of age, in Example 5 they describe a therapeutic effect in 5XFAD mice at 11 and 12 months of age - an extremely progressive stage of beta-amyloid deposition plaques and cognitive failure in this model. Therefore, it is expected that the proposed invention will be relevant for patients at different stages of disease progression, such as Stage 1 - mild/early (lasts 2-4 years); Stage 2 - moderate/medium (lasts 2-10 years); and Stage 3 - severe/late (lasts 1-3 years or longer).

[109] As used herein, the term "CNS function" refers to, among other things, the reception and processing of sensory information, thinking, learning, remembering, perception, language production and understanding, control of motor function, auditory and visual response, maintenance of balance and equilibrium, coordination of movement, transmission of sensory information and control of autonomic functions such as breathing, heart rate and digestion.

[110] As used herein, the terms "cognition", "cognitive function" and "cognitive activity" are used interchangeably and refer to any mental process or state that includes, but is not limited to, learning, memory, imaging, thinking, understanding, reasoning, spatial orientation, speech and language, language acquisition, and voluntary attention potential. Cognition is formed in several areas of the brain, such as the hippocampus, cortex, and other brain structures. However, it is believed that long-term memories are stored, at least in part, in the cortex, and it is also known that sensory information is acquired, integrated and retrieved by a specific cortical structure, the gustatory cortex, which is located within the insular cortex.

[111] In humans, cognitive function can be measured using any known method, such as, but not limited to, the Clinical Global Impression Scale (CIBIC-plus); Mini-Mental State Examination (MMSE); Neuropsychiatric Inventory (NPI); Clinical Dementia Rating Scale (CDR); Cambridge Neuropsychological Test Automated Battery (CANTAB) or Sandoz Clinical Assessment Geriatric Scale (SCAG). Cognitive function can also be measured indirectly using imaging techniques such as positron emission tomography (PET), functional magnetic resonance imaging (fMRI), single photon emission computed tomography (SPECT), or any other imaging technique that measures brain activity.

[112] Improving one or more processes affecting cognition in a patient will mean improving cognitive function in said patient, so in some embodiments, improving cognition includes improving learning, plasticity, and/or long-term memory. The terms "improvement" and "promotion" can be used interchangeably.

[113] The term “learning” refers to the acquisition of new or changing and consolidating existing knowledge, behavior patterns, skills, values or preferences.

[114] The term plasticity refers to synaptic plasticity, brain plasticity, or neuroplasticity, related to the brain's ability to change through learning and modify already acquired memory. One measurable parameter that reflects plasticity is memory extinction.

[115] The term memory refers to the process by which information is encoded, stored, and retrieved. Memory has three different categories: sensory memory, short-term memory and long-term memory.

[116] The term long-term memory refers to the ability to retain information for long or unlimited periods of time. Long-term memory consists of two main sections: explicit memory (declarative memory) and implicit memory (hidden memory). Long-term memory is achieved through memory consolidation, which is a category of processes that stabilize a memory trace after initial acquisition. Consolidation is divided into two specific processes: synaptic consolidation, which occurs within the first few hours after learning, and systemic consolidation, in which hippocampus-dependent memories become hippocampus-independent over a period of weeks to years.

[117] The above embodiments describing various features of the pharmaceutical composition of the present invention are also relevant to the method of the invention since the same pharmaceutical composition is used in this method.

[118] In yet another aspect, the present invention provides a technique for reducing the burden of Aβ plaques in a patient diagnosed with Alzheimer's disease, comprising administering to said patient an active agent or pharmaceutical composition as defined above, thereby reducing the level of systemic immunosuppression by removing the restriction , imposed on the immune system by one or more immune checkpoints.

[119] In yet another aspect, the present invention provides a method of reducing hippocampal gliosis in a patient diagnosed with Alzheimer's disease, comprising administering to said patient an active agent or pharmaceutical composition as described above, which results in a decrease in the level of systemic immunosuppression by removing the restriction imposed by on the immune system by one or more immune checkpoints.

[120] Pharmaceutical compositions for use in accordance with the present invention can be prepared in conventional manner using one or more physiologically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the drug and must not be harmful to the recipient.

[121] The following are examples of carriers, routes of administration, dosage forms, etc. are listed as known possibilities from which carriers, routes of administration, dosage forms, etc. can be selected. for use with the present invention. Those skilled in the art will, however, appreciate that each dosage form and route of administration selected must first be tested to determine whether the desired results are achieved.

[122] Routes of administration include, but are not limited to, parenteral (eg, intravenous, intraperitoneal, intramuscular, subcutaneous), mucosal (eg, oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical, and intradermal routes. Administration may be systemic or local.

[123] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle by which the active agent is administered. The carriers in the pharmaceutical composition may contain a binder such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent such as alginic acid, corn starch and the like; a lubricant or surfactant such as magnesium stearate or sodium lauryl sulfate; as well as a glidant such as colloidal silica.

[124] For oral administration, the pharmaceutical composition may be in liquid form, such as solutions, syrups or suspensions, or may be formulated for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (for example, lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters or fractionated vegetable oils); and preservatives (for example, methyl, propyl p-hydroxybenzoates or sorbic acid). Pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional methods, with pharmaceutically acceptable auxiliaries such as binding agents (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silicon dioxide); leavening agents (for example, potato starch or sodium starch glycolate); or wetting agents (eg sodium lauryl sulfate). Tablets can be coated by methods well known in the art.

[125] Oral formulations can be suitably formulated to obtain controlled release of the active ingredient.

[126] For buccal (buccal) administration, the compositions may take the form of tablets or lozenges, with a conventional formulation.

[127] The compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, such as ampoules or multi-dose containers, with the addition of a preservative. The compositions may take forms such as suspensions, solutions or emulsions in oily or aqueous bases, and may contain excipients such as suspending, stabilizing and/or dispersing agents. In addition, the active ingredient may be in powder form for association with a suitable carrier, for example, sterile pyrogen-free water, before use.

[128] The compositions may also be formulated in rectal formulations such as suppositories or retention enemas, for example containing conventional suppository bases such as cocoa butter or other glycerides.

[129] For administration by inhalation, compositions for use in accordance with the present invention are delivered in a convenient form as an aerosol in pressurized packs or a nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by installing a valve to administer the metered amount. Capsules and cartridges, for example, gelatin for use in an inhaler or insufflator, can be formulated using a powder mixture of the drug and a suitable powder base such as lactose or starch.

[130] Determination of doses of the active ingredient used in the treatment of humans is based on generally accepted practices in the art and should be ultimately determined by physicians in clinical trials. The expected approximate equivalent dose for human administration can be calculated based on the in vivo experimental data described herein below using known formulas (eg, Reagan-Show et al. (2007) Dosetranslation from animals to human studies revisited. TheFASEB Journal 22:659-661). Under this paradigm, the adult equivalent human dose (mg/kg body weight) is equal to the mouse dose (mg/kg body weight) multiplied by 0.081.

[131] Aspects of the present specification may also be summarized as follows:

1. A method of treating a disease, disorder, pathological condition or lesion of the central nervous system in an individual in need thereof, the method comprising administering to the individual a composition containing an active agent that causes a decrease in the level of systemic immunosuppression by removing the restriction imposed on the immune system by one or more immune checkpoints, wherein the composition is administered in a dosage regimen comprising at least one course of treatment, each course of treatment comprising sequentially a treatment session in which the composition is administered to the individual, followed by a no-treatment session in which the composition is not administered to the individual, wherein a period without treatment is longer than the treatment session; wherein, if the administration of the composition during a treatment session is repeated, the period without treatment will be longer than the period between repeated administrations during a treatment session; wherein administration of the composition transiently reduces levels of systemic immunosuppression and increases the gating activity of the choroid plexus in facilitating the selective recruitment of immune cells into the central nervous system, thereby providing treatment to the individual.

2. An active agent or a pharmaceutical composition containing an active agent for use in the treatment of a disease, disorder, condition or lesion of the central nervous system, where the active agent causes a decrease in the level of systemic immunosuppression by removing the restriction imposed on the immune system by one or more immune checkpoints wherein the active agent or pharmaceutical composition is administered through a dosage regimen comprising at least one course of treatment, wherein each course of treatment comprises sequentially a treatment session in which the composition is administered to the individual, followed by a non-treatment session in which the composition is not administered to the individual, wherein the period without treatment is longer than the treatment session; wherein, if the administration of the composition during a treatment session is repeated, the period without treatment will be longer than the period between repeated administrations during a treatment session; wherein administration of the composition transiently reduces levels of systemic immunosuppression and increases the gating activity of the choroid plexus in facilitating the selective recruitment of immune cells into the central nervous system, thereby providing treatment to the individual.

3. The use of an active agent that reduces the level of systemic immunosuppression by removing the restrictions imposed on the immune system by one or more immune checkpoints to treat a disease, disorder, pathological condition or lesion of the central nervous system.

4. The use of an active agent that reduces the level of systemic immunosuppression by removing restrictions imposed on the immune system by one or more immune checkpoints in the manufacture of a medicinal product for the treatment of a disease, disorder, condition or lesion of the central nervous system.

5. The method of Embodiment 1, the active agent or pharmaceutical composition of Embodiment 2, or the use of Embodiment 3 or 4, wherein the active agent is an antibody, antibody mimic, aptamer, small molecule, neem leaf glycoprotein, sCTLA-4 , a silencing molecule, OK-432, a combination of IL-12 and anti-CTLA-4, or any combination of the above.

6. The method of Embodiment 5, the active agent or pharmaceutical composition of Embodiment 5, or the use of Embodiment 5, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

7. The method of embodiments 5 or 6, the active agent or pharmaceutical composition of embodiments 5 or 6, or the use of embodiments 5 or 6, wherein the antibody is a dimer, multimer, multispecific antibody, recombinant antibody, chimeric antibody, bifunctional antibody , a cell-associated antibody, such as an Ig receptor, linear antibody, diabody, minibody, or nanobody.

8. The method of any one of embodiments 5-7, the active agent or pharmaceutical composition of embodiments 5-7, or the use of embodiment 5-7, wherein the antibody is a human or humanized antibody.

9. The method of any one of embodiments 5-8, the active agent or pharmaceutical composition of embodiments 5-8, or the use of embodiment 5-8, wherein the antibody is an antagonist antibody or an agonist antibody.

10. The method of any one of embodiments 5-9, the active agent or pharmaceutical composition of embodiments 5-9, or the use of embodiment 5-9, wherein the antibody is a neutralizing antibody.

11. The method of any one of embodiments 5-10, the active agent or pharmaceutical composition of embodiments 5-10, or the use of embodiment 5-10, wherein the antibody is a full-length immunoglobulin molecule or an immunologically active fragment.

12. The method of embodiment 11, the active agent or pharmaceutical composition of embodiment 11, or the use of embodiment 11, wherein the immunologically active fragment is a single domain antibody (sdAb), a single chain variable fragment (scFv), a Fab fragment, F(ab')2 fragment, Fc fragment, Fd fragment, Fv fragment.

13. The method of embodiment 5-12, the active agent or pharmaceutical composition of embodiment 3 or 4, or the use of any of embodiments 4-11, wherein the antibody is anti-PD-1, anti-PDL1, anti- PDL2, anti-CTLA-3, anti-CD80, anti-CD86, anti-B7RP1, anti-B7-H3, anti-B7-H4, anti-B7-H7, anti-BTLA, anti-HVEM, anti-CD- 4, anti-CD40, anti-CD40L, anti-CD70, anti-CD80, anti-CD86, anti-CD137, anti-CDI 37L, anti-OX40, anti-OX40L anti-TIM-4-11, anti-Galectin9, anti-KIR, anti-LAG-1, anti-ICOS, anti-VISTA, anti-STING, anti-TIGIT, anti-GITR antibody or any combination thereof.

14. The method of Embodiment 5, the active agent or pharmaceutical composition of Embodiment 5, or the use of Embodiment 5, wherein the antibody mimetic is an affinity molecule, affilin, affimer, affitin, alpha, anticalin, avimer, darpin , finomer, Kunitz domain peptide or monobody.

15. The method of Embodiment 5, the active agent or pharmaceutical composition of Embodiment 5, or the use of Embodiment 4, wherein the aptamer is a DNA aptamer, an RNA aptamer, a XNA aptamer, or a peptide aptamer.

16. The method of Embodiment 5, the active agent or pharmaceutical composition of Embodiment 5, or the use of Embodiment 5, wherein the small molecule is a p300 inhibitor, sunitinib, polyoxometalate-1, α,β-methylenadenosine 5'-diphosphate, arsenic trioxide, GX15-070, retinoic acid antagonist, CCR4 antagonist, adenosine receptor antagonist,

adenosine A1 receptor antagonist; adenosine A2a receptor, adenosine A2b receptor antagonist, A3 receptor antagonist, indoleamine 2,3-dioxygenase antagonist or HIF-1 regulator.

17. The method of any one of embodiments 1-16, the active agent or pharmaceutical composition of embodiments 2-16, or the use of embodiment 3-16, wherein administration of the composition during a treatment session is a single administration.

18. The method of any one of embodiments 1-16, the active agent or pharmaceutical composition of embodiments 2-16, or the use of embodiment 3-16, wherein the administration of the composition during a treatment session is a repeated administration.

19. The method of embodiment 18, the active agent or pharmaceutical composition of embodiment 18, or the use of embodiment 18, wherein repeated administration is performed once every day, once every two days, once every three days, once every four days, once every five days or every six days.

20. The method of embodiment 18, the active agent or pharmaceutical composition of embodiment 18, or the use of embodiment 18, wherein repeated administration is performed once every week, every two weeks, every three weeks, or every four weeks.

21. The method of any one of embodiments 1-20, the active agent or pharmaceutical composition of embodiments 2-20, or the use of embodiment 3-20, wherein the treatment session takes from 1 day to four weeks.

22. The method of embodiment 21, the active agent or pharmaceutical composition of embodiment 21, or the use of embodiment 21, wherein the treatment session lasts from 3 days to four weeks.

23. The method of embodiment 22, the active agent or pharmaceutical composition of embodiment 22, or the use of embodiment 22, wherein the treatment session takes from one week to four weeks.

24. The method of any one of embodiments 1-23, the active agent or pharmaceutical composition of embodiments 2-23, or the use of embodiment 3-23, wherein the treatment-free period is from one week to six months.

25. The method of embodiment 24, the active agent or pharmaceutical composition of embodiment 24, or the use of embodiment 24, wherein the treatment-free period is from two weeks to six months.

26. The method of embodiment 25, the active agent or pharmaceutical composition of embodiment 25, or the use of embodiment 25, wherein the treatment-free period is three weeks to six months.

27. The method of embodiment 26, the active agent or pharmaceutical composition of embodiment 26, or the use of embodiment 26, wherein the treatment-free period is one to three months.

28. The method of embodiment 27, the active agent or pharmaceutical composition of embodiment 27, or the use of embodiment 27, wherein the treatment-free period is one to two months.

29. The method of any one of embodiments 1-28, the active agent or pharmaceutical composition of any of embodiments 2-28, or the use of any of embodiments 3-28, wherein a decrease in the level of systemic immunosuppression is associated with an increase in systemic presence or activity IFNγ-producing leukocytes and/or an increase in the systemic presence or activity of the cytokine IFNγ.

30. The method of any of embodiments 1-29, the active agent or pharmaceutical composition of any of embodiments 2-29, or the use of any of embodiments 3-29, wherein the transient reduction in the level of systemic immunosuppression is associated with an increase in the systemic presence or effector T cell activity.

31. The method of any of embodiments 1-30, the active agent or pharmaceutical composition of any of embodiments 2-30, or the use of any of embodiments 3-30, wherein the temporary reduction in the level of systemic immunosuppression is associated with a decrease in the systemic presence or activity of regulatory T cells and/or a decrease in the systemic presence of the cytokine IL-10.

32. The method of any one of embodiments 1-31, the active agent or pharmaceutical composition of any of embodiments 2-31, or the use of any of embodiments 3-31, wherein the transient reduction in the level of systemic immunosuppression is associated with a decrease in the systemic presence of suppressive drugs myeloid-derived cells (MDSC).

33. The method of any one of embodiments 1-32, the active agent or pharmaceutical composition of any of embodiments 2-32, or the use of any of embodiments 3-32, wherein the temporary reduction in the level of systemic immunosuppression occurs by removing the restriction imposed on the immune system by one or more immune checkpoints.

34. The method of embodiment 33, the active agent or pharmaceutical composition of embodiment 33, or the use of embodiment 33, wherein administration of the composition blocks one or more immune checkpoints, thereby causing a temporary reduction in the level of systemic immunosuppression.

35. The method of embodiment 34, the active agent or pharmaceutical composition of embodiment 34, or the use of embodiment 34, wherein the one or more immune checkpoints include PD-1-PDL1, PD-1-PDL2, CD28-CD80, CD28-CD86, CTLA-4-CD80, CTLA-4-CD86, ICOS-B7RP1, B7H3, B7H4, B7H7, B7-CD28-like molecule, BTLA-HVEM, KIR-MHC class I or II, LAG3-MHC class I or II, CD137-CD137L, OX40-OX40L, CD27-CD70, CD40L-CD40, TIM3-GAL9, V-domain immunoglobulin suppressor of T-cell activation (VISTA), stimulator of interferon genes (STING), T-cell immunoglobulin and immunoreceptor inhibitor tyrosine-based domain motif (TIGIT), glucocorticoid-induced tumor necrosis factor-related protein (GITR), A2aR-adenosine or indoleamine 2,3-dioxygenase (IDO)-L-tryptophan.

36. The method of any one of embodiments 1-35, the active agent or pharmaceutical composition of any of embodiments 2-35, or the use of any of embodiments 3-35, wherein administration of the composition during the treatment session continues for at least until until the systemic presence or activity of IFNγ-producing leukocytes and/or IFNγ cytokine is higher than control, after which the administration is stopped and the treatment-free period continues until the systemic presence or activity of IFNγ-producing leukocytes and/or the IFNγ cytokine level remains above control, where control includes: a) the level of systemic presence or activity of IFNγ-producing leukocytes and/or IFNγ cytokine measured in the last blood sample obtained from the individual prior to administration; or b) the level of systemic presence or activity of IFNγ-producing leukocytes and/or the cytokine IFNγ characteristic of a group of individuals suffering from a disease, disorder, condition or lesion of the central nervous system.

37. The method of any one of embodiments 1-36, the active agent or pharmaceutical composition of any of embodiments 2-36, or the use of any of embodiments 3-36, wherein the individual reduces the level of soluble amyloid beta peptide in the brain , reduced or decreased brain amyloid beta (Aβ) plaque burden, decreased hippocampal gliosis, decreased proinflammatory cytokine levels in the brain, decreased brain inflammation, and/or improved cognitive function.

38. The method of Embodiment 37, the active agent or pharmaceutical composition of Embodiment 37, or the use of Embodiment 37, wherein the improved cognitive function is learning, memory, imaging, plasticity, reasoning, awareness, reasoning, spatial ability perception, speech and language skills, language acquisition, judgmental ability, or any combination of these.

39. The method of any one of embodiments 1-38, the active agent or pharmaceutical composition of embodiments 2-38, or the use of embodiment 3-38, wherein the immune cells include monocytes, macrophages or T cells.

40. The method of embodiment 39, the active agent or pharmaceutical composition of embodiment 39, or the use of embodiment 39, wherein the T cells include regulatory T cells.

41. The method of any one of embodiments 1-40, the active agent or pharmaceutical composition of any of embodiments 2-40, or the use of any of embodiments 3-40, wherein the diseases, disorders, pathological conditions or lesions of the central nervous system include Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis; secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, anterior ischemic optic neuropathy; glaucoma; uveitis; depression; Trauma-related stress or post-traumatic stress disorder, frontotemporal dementia, diffuse Lewy body disease, mild cognitive impairment, posterior cortical atrophy, primary progressive aphasia, or progressive supranuclear palsy.

42. The method of any one of embodiments 1-40, the active agent or pharmaceutical composition of any of embodiments 2-40, or the use of any of embodiments 1-40, wherein the disease, disorder, pathological condition or lesion of the central nervous system is tauopathy.

43. The method of Embodiment 42, the active agent or pharmaceutical composition of Embodiment 42, or the use of Embodiment 42, wherein the tauopathy is Alzheimer's disease, argyrophilic grain disease, chronic traumatic encephalopathy, corticobasal degeneration, boxer's dementia, frontotemporal dementia , frontotemporal degeneration, Hallerwarden-Spatz disease, Huntington disease, ganglioglioma, gangliocytoma, globular glial tauopathy, lead encephalopathy, lipofuscinosis, Lytico-Bodig disease (Parkinson's disease and dementia complex in Guam), meningoangiomatosis, parkinsonism associated with chromosome 17, Pick's disease, primary age-related tauopathy (PART), formerly known as dementia with neurofibrillary tangles (NFT dementia), postencephalitic parkinsonism, progressive supranuclear palsy, subacute sclerosing panencephalitis or tuberous sclerosis.

44. The method of any one of embodiments 1-40, the active agent or pharmaceutical composition of any of embodiments 2-40, or the use of any of embodiments 3-40, wherein the disease, disorder, condition, or lesion of the central nervous system is retinal degeneration.

45. The method of Embodiment 44, the active agent or pharmaceutical composition of Embodiment 44, or the use of Embodiment 44, wherein the retinal degeneration is wet age-related macular degeneration, dry age-related macular degeneration, retinitis pigmentosa, choroideremia, cone-rod retinal degeneration , gyrate atrophy, juvenile retinoschisis, icteric macular degeneration (Best disease), abetalipoproteinemia (Bassen-Kornzweig disease), Bardet-Biedl syndrome, blue cone syndrome, dominant drusen, Goldmann-Favre vitreoretinal dystrophy (enhanced S-cone syndrome), syndrome Kearns-Sayre syndrome, Lawrence-Moon syndrome, Leber congenital amaurosis, Refsum Leber disease, Oguchi disease, peripapillary (pericentral) choroidal dystrophy, pigmentary dystrophy, Sorsby macular dystrophy, Stargardt disease, Stickler syndrome, Usher syndrome or Wagner vitreoretinal dystrophy.

EXAMPLES

[132] The following examples, without limitation, are presented for illustrative purposes only to facilitate a more complete understanding of the representative embodiments contemplated. These examples should not be construed as limiting any of the embodiments presented herein, including those relating to the active agents, pharmaceutical compositions, or methods and uses disclosed herein.

Materials and methods

[133] Animals. 5XFAD transgenic mice (Tg6799) overexpressing familial AD mutant forms of human APP (Swedish mutation, K670N/M671L; Florida mutation, I716V; and London mutation, V717I), PS1 (M146L/L286V) transgenes under transcriptional control of a neuron-specific catalyst for mice Thy-1 (Oakley et al, 2006) and AD B6 double transgenic mice. Cg-Tg (APPswe, PSEN1dE9) 85Dbo/J (Borchelt et al, 1997) were purchased from the Jackson Laboratory. Genotyping was performed by PCR analysis of tail DNA as described above (Oakley et al, 2006). Heterozygous mutant cx ₃ cr1 ^GFP/+ mice (Jung et al, 2000) (B6.129P-cx3cr1 ^tm1Litt /J, where one of the CX ₃ CR1 chemokine receptor alleles is replaced by a gene encoding GFP) were used as donors for bone marrow chimeras. Foxp3.LuciDTR mice were bred from 5XFAD mice to activate conditional depletion of Foxp3 ⁺ Treg cells. The animals were bred and maintained by the Weizmann Institute Animal Breeding Center. All experiments described herein were conducted in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute of Science.

[134] RNA purification, cDNA synthesis and quantitative real-time PCR analysis. Total hippocampal dentate gyrus (DG) RNA was extracted with TRI Reagent (Molecular Research Center) and purified from lysates using the RNeasy kit (Qiagen). Total choroid plexus RNA was prepared using the MicroPrep RNA kit (Zymo Research). mRNA (1 μg) was converted to cDNA using a high-throughput cDNA reverse transcriptase kit (Applied Biosystems). Expression of specific mRNAs was analyzed using fluorescent real-time quantitative PCR (RT-qPCR). RT-qPCR reactions were performed using Fast-SYBR PCM Master Mix (Applied Biosystems). Quantification reactions were performed in triplicate for each sample using the standard curve method. The peptidyl-prolyl isomerase A (ppia) gene was chosen as the reference gene. Amplification cycles were 95°C for 5 s, 60°C for 20 s, and 72°C for 15 s. At the end of the assay, a melting curve was generated to assess the specificity of the reaction. For analysis of the ifn-γ and ppia genes, cDNA was preamplified by 14 cycles of PCR with nonrandom PCR primers, thereby increasing the sensitivity of subsequent real-time PCR analysis according to the manufacturer's protocol (PreAmp Master Mix Kit; Applied Biosystems). 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 V2.2.2 software (Applied Biosystems). The following TaqMan Assays-on-Demand™ probes were used: Mm02342430_g1 (ppia) and Mm01168134_m1 (ifn-γ).

[135] For all other genes studied, the following primers were used:

[136] ppia forward 5'-AGCATACAGGTCCTGGCATCTTGT-3' (ID number: 33) and reverse 5'-CAAAGACCACATGCTTGCCATCCA-3' (ID number: 34);

[137] icam1 forward 5'-AGATCACATTCACGGTGCTGGCTA-3' (ID number: 35) and reverse 5'-AGCTTTGGGATGGTAGCTGGAAGA-3' (ID number: 36);

[138] vcam1 forward 5'-TGTGAAGGGATTAACGAGGCTGGA-3' (ID number: 37) and reverse 5'-CCATGTTTCGGGCACATTTCCCACA-3' (ID number: 38);

[139] cxcl10 forward 5'-AACTGCATCCATATCGATGAC-3' (ID number: 39) and reverse 5'-GTGGCAATGATCTCAACAC-3' (ID number: 40);

[140] ccl2 forward 5'-CATCCACGTGTTGGCTCA-3' (ID number: 41) and reverse 5'-GATCATCTTGCTGGTGAATGAGT-3' (ID number: 42);

[141] tnf-γ forward 5'-GCCTCTTCTCATTCCTGCTT-3' (ID number: 43) reverse CTCCTCCACTTGGTGGTTTG-3' (ID number: 44);

[142] il-1β forward 5'-CCAAAAGATGAAGGGCTGCTT-3' (ID: 45) and reverse 5'-TGCTGCTGCGAGATTTGAAG-3' (ID: 46);

[143] il-12p40 forward 5'-GAAGTTCAACATCAAGAGCA-3' (ID number: 47) and reverse 5'-CATAGTCCCTTTGGTCCAG-3' (ID number: 48);

[144] il-10 forward 5'-TGAATTCCCTGGGTGAGAAGCTGA-3' (ID number: 49) and reverse 5'-TGGCCTTGTAGACACCTTGGTCTT-3' (ID number: 50);

[145] tgfβ2 forward 5'-AATTGCTGCCTTCGCCCTCTTTAC-3' (ID number: 51) and reverse 5'-TGTACAGGCTGAGGACTTTGGTGT-3' (ID number: 52);

[146] igf-1 forward 5'-CCGGACCAGAGACCCTTTG (ID number: 53) and reverse 5'-CCTGTGGGCTTGTTGAAGTAAAA-3' (ID number: 54);

[147] bdnf forward 5'-GATGCTCAGCAGTCAAGTGCCTTT-3' (ID number: 55) and reverse 5'-GACATGTTTGCGGCATCCAGGTAA-3' (ID number: 56);

[148] Immunohistochemistry. Tissue processing and immunohistochemistry were performed on paraffin sections of mouse (6 μm thick) and human (10 μm thick) brains. Mouse ICAM primary antibody (1:20 Abcam, ab2213) was used to stain human ICAM-1. Slides were incubated for 10 min in 3% H2O2, and a secondary biotin-conjugated anti-mouse antibody was used, followed by biotin/avidin amplification using the Vectastain ABC kit (Vector Laboratories). 3,3'-Diaminobenzidine (DAB substrate) was then used (Zytomed kit); the slides were dehydrated and mounted using a xylene-based mounting solution. For tissue staining, mice were transcardially perfused with PBS until tissue was excised and fixed. CC tissues were separated under a dissecting microscope (Stemi DV4; Zeiss) from the lateral, third, and fourth ventricles of the brain. For CC whole compound staining, tissues were fixed with 2.5% paraformaldehyde (PFA) for 1 hour at 4°C and then transferred to PBS containing 0.05% sodium azide. Before staining, dissected tissues were washed with PBS and blocked (20% horse serum, 0.3% Triton X-100, and PBS) for 1 hour at room temperature. Staining with complete primary antibody (in PBS containing 2% horse serum and 0.3% Triton X-100) or with secondary antibodies was carried out for 1 hour at room temperature. Each step was followed by three washes in PBS. Tissues were mounted on slides, mounted using an Immu-mount (9990402, Thermo Scientific), and sealed using a cover. Two different tissue preparation protocols (paraffin embedded or microtome free sections) were used to stain brain sections 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-Cahedrin, and rabbit anti-Claudin-1 (all 1:100, Invitrogen, #33-9100, #33-4000, #51-9000); rabbit anti-GFKB (1:200, Dako, #Z0334). Secondary antibodies included donkey anti-mouse/goat/rabbit/rat Cy2/Cy3/Cy5 conjugated antibodies (1:200, all from Jackson Immunoresearch). Slides were subjected to Hoechst nuclear staining (1 4000; Invitrogen Probes) for 1 min. In immunostaining procedures, two negative controls were routinely used, staining with an isotype control antibody followed by staining with a secondary antibody, or staining with the secondary antibody alone. For intracellular Foxp3 staining, antigen retrieval from paraffin slides was performed using the Retreivagen Kit (#550524, #550527, BD Pharmingen™). 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 a 20× NA 0.50 or 40× NA 0.75 objective (Plan Fluor, Nikon). The confocal microscope was equipped with an LSM 510 laser scanning device (three lasers: Ar 488, HeNe 543 and HeNe 633). Recordings were made on secondary fixed tissues using capture software (NIS-Elements, F3 [Nikon] or LSM [Carl Zeiss, Inc.]). To quantify staining intensity, total cell and background staining was measured using ImageJ software (NIH), and specific staining intensity was calculated as described previously (Burgess et al, 2010). Images were cropped, merged and optimized using Photoshop CS6 13.0 (Adobe), and organized using Illustrator CS5 15.1 (Adobe).

[149] Paraffin sections of human CC. Postmortem brain sections from young and elderly non-CNS patients and patients with AD were obtained from the Oxford Brain Bank (formerly known as the Thomas Willis Oxford Brain Collection (TWOBC)) with appropriate consent and approval from the Ethics Committee ( TW220). Experiments using these sections were approved by the Weizmann Institute of Bioethics Committee.

[150] Flow cytometry, sample preparation and analysis. Mice were transcardially perfused with PBS and tissues were processed as described previously (Baruch et al, 2013). Brains were dissected, various brain sections were removed under a dissecting microscope (Stemi DV4; Zeiss) in PBS, and tissues were separated using a gentleMACS™ dissociator (Miltenyi Biotec). Choroid plexus tissues were isolated from the lateral, third and fourth ventricles of the brain, incubated at 37°C for 45 minutes in PBS (with Ca ²⁺ /Mg ²⁺ ) containing 400 U/ml collagenase type IV (Worthington Biochemical Corporation), and then manually homogenized by pipetting. Spleens were crushed with a syringe plunger and treated with ACK (ammonium chloride potassium) lysis buffer to remove red blood cells. In all cases, samples were stained according to the manufacturers' protocols. All samples were filtered through a 70-μm nylon mesh and blocked with anti-Fc CD16/32 (1:100; BD Biosciences). For intracellular IFN-γ staining, cells were incubated with paramethoxyamphetamine (10 ng/ml, Sigma-Aldrich) and ionomycin (250 ng/ml, Sigma-Aldrich) for 6 h, with Brefeldin-A (10 μg/ml) added for the last 4 h , Sigma-Aldrich). Intracellular cytokine labeling was performed using the BD Cytofix/Cytoperm™ Plus Fixation/Permealization Kit (Cat. No. 555028). The eBioscience FoxP3 Buffer Staining Kit (cat. no. 00-5523-00) was used to stain regulatory T cells. The following fluorochrome-labeled monoclonal antibodies were purchased from BD Pharmingen, BioLegend, R&D Systems, or eBiosciences, and used according to the manufacturers' protocols: PE or Alexa Fluor 450-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; bright purple conjugated anti-CD45. Cells were analyzed on an LSRII cytometer (BD Biosciences) using FlowJo software. Each experiment used appropriate negative controls, positive controls, and single stains for each tissue to identify populations of interest and to exclude other populations.

[151] Preparation of bone marrow (BM) chimeras. Bone marrow chimeras were prepared as described previously (Shechter et al, 2009; Shechter et al, 2013). Briefly, sex-matched recipient mice were exposed to lethal whole-body irradiation (950 rad) with head protection (Shechter et al, 2009). Mice were then injected intravenously with 5 x 10 ⁶ BM cells from CX ₃ CR1 ^GFP/+ donors. Mice were kept for 8–10 weeks after BM cell transplantation to allow restoration of the hematopoietic lineage before they were used in experiments. The percentage of chimerism was determined by FACS analysis of blood samples according to the percentage of GFP-expressing cells among circulating monocytes (CD11b ⁺ ). In this brain-protecting model, an average of 60% chimerism was achieved and CNS-infiltrating GFP ⁺ myeloid cells were confirmed to be CD45 ^high /CD11b ^high , representing monocyte-derived macrophages rather than microglia (Shechter et al, 2013) .

[152] Morris's water maze. Mice were tested three times a day for 4 consecutive days to train them to find a hidden platform located 1.5 cm below the surface of the water in a pool (1.1 m in diameter). The water temperature was maintained at 21-22°C. The water was made opaque using milk powder. In the testing room, mice were presented only with a distant visual shape and cue objects to search for the submerged platform. Rescue latency, i.e. the time required to find the platform and climb onto it was recorded up to 60 s. Each mouse was allowed to remain on the platform for 15 s before being removed from the maze and returned to its cage. If the mouse did not find the platform within 60 s, it was manually placed on the platform and returned to its cage after 15 s. The interval between trials for each mouse was 10 min. On day 5, the platform was removed and mice were subjected to one trial lasting 60 s without the opportunity to find the exit. On days 6 and 7, the platform was placed in the sector opposite the initial training sector, and the mouse was retrained for three sessions each day. Data were recorded using an automated tracking system EthoVision V7.1 (Noldus Information Technology). Statistical analysis was performed using analysis of variance (ANOVA) and Bonferroni post hoc analysis. All tests in the VLM were conducted from 10 am to 5 pm during the lights-off phase.

[153] Radial arm water labyrinth. To test spatial learning and memory, a radial arm water maze (RAWM) was used as previously described in detail (Alamed et al, 2006). Briefly, six stainless steel inserts were placed in the tank to form six floating arms extending from an open central area. The exit platform was located at the end of one of the arms (target arm), 1.5 cm below the water surface in a 1.1 m diameter pool. The water temperature was maintained at 21-22°C. The water was made opaque using milk powder. In the testing room, mice were presented only with a distant visual shape and cue objects to search for the submerged platform. The location of the target arm remained constant for a given mouse. On day 1, mice were subjected to 15 trials (over 3 hours), alternating between visible and hidden platforms, and the last 4 trials with hidden platforms only. On day 2, mice were trained through 15 hidden platform trials. Entering the wrong arm or failing to select an arm within 15 s was recorded as an error. Spatial learning and memory were measured by counting the number of sleeve entry errors or escape latency of mice on each trial. Training data were analyzed as mean escape errors or latencies across blocks of three consecutive trials.

[154] GA Introduction. Each mouse was subcutaneously administered GA at a total dose of 100 μg (Lot No. P53640, Teva Pharmaceutical Industries, Petah Tikva, Israel) dissolved in 200 μl PBS. Mice were injected on a schedule of either weekly GA (Butovsky et al, 2006) or daily GA (Figure 8 and Figure 16). Mice were sacrificed 1 week after the last GA injection, or 1 month after treatment, as indicated for each experiment.

[155] Conditional ablation of regulatory T cells. Diphtheria toxin (DTx, 8 ng/g body weight, Sigma) was administered intraperitoneally daily for 4 consecutive days to Foxp3.LuciDTR mice (Suffner et al, 2010). The efficacy of DTx was confirmed by flow cytometric analysis of immune cells in the blood and spleen, and almost complete (>99%) depletion of GFP-expressing FoxP3 ⁺ CD4 ⁺ Treg cells was achieved (Fig. 4).

[156] 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 administered intraperitoneally (8.9 mg kg ^-1 d ^-1 ) daily for 1 week. Similarly, vehicle-treated mice were administered DMSO.

[157] Treatment of ATGM. Polytransretinoic acid (PTRA) was administered to mice in a similar manner to a previously described procedure (Walsh et al, 2014). PTRK (Sigma) was dissolved in DMSO and administered intraperitoneally. (8 mg kg ^-1 d ^-1 ) every other day for 1 week. Similarly, vehicle-treated mice were administered DMSO.

[158] Isolation and quantification of soluble Aβ protein (sAβ). Tissue homogenization and sAβ protein extraction were performed as described previously (Schmidt et al, 2005) ENREF 54 ENREF 53. Briefly, brain parenchyma was separated, frozen and kept at -80°C until homogenized. Proteins were sequentially extracted from samples to obtain separate fractions containing proteins with different solubilities. Samples were homogenized 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) using a ground glass pestle in a Dounce homogenizer. After six strokes, the homogenate was mixed 1:1 with 0.4% diethylamine (DEA) in 100 mM NaCl solution before another six strokes, and then centrifuged at 135,000 g at 4°C for 45 minutes. The supernatant (DEA-soluble fraction containing extracellular and cytosolic proteins) was collected and neutralized with 10% 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, respectively) according to the manufacturer's instructions.

[159] Quantitative determination of Aβ plaques. 6-μm coronal sections were collected from each brain sample, and eight sections per mouse, from four different predefined depths throughout the region of interest (dentate gyrus or cerebral cortex), were immunostained. Histogram-based segmentation of positively colored 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 or cortical layer 5, and the percentage of area occupied by complete Aβ immunostaining was determined. The number of plaques was counted on the same 6 μm coronal brain sections and presented as the average number of plaques per brain region. Prior to quantification, sections were coded to conceal experimental group identifiers, and plaque burden was calculated by an observer blinded to group identification.

[160] Statistical analysis. The specific tests used to analyze each set of experiments are indicated in the figure legends. Data were analyzed using a two-tailed Student's t test for comparisons between two groups, one-way ANOVA for comparisons across multiple groups, followed by Newman-Keuls post-hoc analysis for pairwise comparisons of groups after rejecting the null hypothesis (P<0 .05). Behavioral test data were analyzed using two-way repeated measures ANOVA, and post hoc analysis with Bonferroni's test for pairwise comparisons. Sample sizes were selected with sufficient statistical power based on the literature and past experience, and mice were allocated to experimental groups based on age, sex, and genotype. During the experiments and when assessing the results, the researchers could not identify the groups (“blind method”). All inclusion and exclusion criteria were pre-established according to IACUC guidelines. Results are presented as mean ± standard error of the mean. In the graphs, the y-axis represents the standard error of the mean. Statistical calculations were performed using GraphPad Prism software (GraphPad Software, San Diego, CA).

Example 1. "Gateway" activity of the choroid plexus (CP) during disease progression in a mouse model of AD.

[161] We first examined the activity of CP during disease progression in the 5XFAD transgenic mouse model (AD-Tg); these mice coexpress five mutations associated with familial AD and develop cerebral Aβ pathology and gliosis as early as 2 months of age (Oakley et al, 2006). We found that during progressive stages of disease pathology, CP AD-Tg mice, compared to age-matched wild-type (WT) controls, expressed significantly lower levels of leukocyte homing and migration determinants, including icam1, vcam1, cxcl10 and ccl2 (Figure 1A), showing up-regulation by CP in response to acute CNS injury, and required transepithelial migration of leukocytes (Kunis et al, 2013; Shechter et al, 2013). Immunohistochemical staining for the integrin ligand, ICAM-1, confirmed its reduced expression by the CP epithelium of AD-Tg mice (Fig. 1b). Additionally, staining for ICAM-1 in postmortem human brains showed an age-related decrease in CP epithelium, consistent with our previous observations (Baruch et al, 2014), and quantification of this effect revealed a further decrease in AD patients compared with elderly people without CNS diseases (Fig. 2A). Because induction of leukocyte migration determinants by the CP is dependent on epithelial interferon (IFN)-γ signaling (Kunis et al, 2013), we next tested whether the observed effects reflected a loss of IFN-γ availability in the CP. Examination of the CP of 5XFAD AD-Tg mice using intracellular flow cytometry staining revealed significantly fewer IFN-γ-producing cells in this compartment (Fig. 2B), and quantitative real-time PCR (RT-qPCR) analysis confirmed lower levels of expression ifn-γ mRNA in the CP of AD-Tg mice compared with age-matched wild-type controls ( Fig. 2C ).

Example 2. Functional connections between Treg-mediated systemic immunosuppression, CP gateway activity and AD pathology.

[162] Regulatory T cells (Treg) play a key role in suppressing systemic effector immune responses (Sakaguchi et al, 2008). We hypothesized that Treg-mediated systemic immunosuppression affects IFN-γ availability in the CP and accordingly focused on the involvement of regulatory T cells in AD pathology. Consistent with previous reports of elevated Treg levels and suppression in AD patients (Rosenkranz et al, 2007; Saresella et al, 2010; Torres et al, 2013), assessing the frequency of Foxp3 ⁺ Tregs in splenocytes of 5XFAD AD-Tg mice compared with their age-matched wild-type littermates showed increased levels with disease progression (Fig. 3A, B). To study the functional links between Treg-mediated systemic immunosuppression, CP gateway activity, and AD pathology, we crossed 5XFAD AD-Tg mice with Foxp3-diphtheria toxin receptor (DTR ⁺ ) mice, which provided a transient in vivo conditional depletion of Foxp3 ⁺ Treg cells in AD-Tg/DTR ⁺ mice by administration of diphtheria toxin (DTx) ENREF 33 (Fig. 4A). Transient depletion of regulatory T cells resulted in increased mRNA expression of leukocyte trafficking molecules by CP AD-Tg/DTR ⁺ mice compared with their DTx-treated AD-Tg/DTR littermates ( Fig. 5A ). Analysis of the long-term effect of temporary depletion of regulatory T cells on the brain parenchyma (3 weeks later) revealed the accumulation of immune cells in the brain, including increased numbers of CD45 ^high /CD11 ^high myeloid cells representing infiltrating mo-MFs (Shechter et al, 2013) and CD4 ⁺ T cells (Fig. 5B). In addition, transient depletion of regulatory T cells resulted in a marked enrichment of Foxp3 ⁺ Treg cells among CD4 ⁺ T cells that accumulated in the brain, as assessed by flow cytometry ( Figures 5C,D ). Real-time quantitative PCR (RT-qPCR) examination of the hippocampus showed increased expression of foxp3 and il10 mRNA ( Figure 5E ).

[163] We then examined whether short-term depletion of regulatory T cells, after which immunoregulatory cells accumulate in areas of brain pathology, leads to long-term effects on brain activity. We observed decreased hippocampal gliosis (Figure 5F) and decreased mRNA expression of proinflammatory cytokines such as il-12p40 and tnf-α (Figure 5G). Additionally, the burden of cerebral Aβ plaques in the dentate gyrus of the hippocampus as well as the cerebral cortex (layer 5), two brain regions showing robust Aβ plaque pathology in 5XFAD AD-Tg mice (Oakley et al, 2006), was reduced (Fig. 6A, B). Assessment of the effect on cognitive function using the Morris water maze (WLM) showed significant improvements in spatial learning and memory in AD-Tg/DTR ⁺ mice after regulatory T cell depletion compared with AD-Tg/DTR mice treated with DTx, achieving performance similar to that of wild-type mice (Fig. 6C-E). Taken together, these data demonstrated that transient disruption of Treg-mediated systemic immunosuppression in AD-Tg mice resulted in the accumulation of anti-inflammatory cells, including mo-MPs and Tregs, in the brain, followed by resolution of the neuroinflammatory response, clearance of Aβ, and reversal of cognitive decline.

Example 3. Weekly administration of copolymer-1 reduces Treg-mediated systemic immunosuppression, increases CP gate activity and mitigates AD pathology.

[164] To further substantiate the causal nature of the inverse relationship between systemic immunosuppression, CV activity, and AD pathology, we then used an immunomodulatory compound, glatiramer acetate (GF, GA, also known as copolymer-1 or Copaxone®), which was assessed by weekly administration demonstrated a therapeutic effect in the APP/PS1 mouse model of AD (Butovsky et al, 2006); ENREF 22 this effect was functionally associated with the recruitment of mo-MF to disease-affected brain regions (Butovsky et al, 2007). In this case, we first examined whether CC in AD-Tg APP/PS1 mice, similar to our observation in 5XFAD AD-Tg mice, was deficient in terms of IFN-γ expression levels. We found that in AD-Tg APP/PS1 mice, IFN-γ d CP levels were reduced compared to age-matched wild-type controls (Fig. 7A). These results prompted the inventors to test whether the therapeutic effect of weekly GA administration in APP/PS1 mice (Butovsky et al, 2006) could be reproduced in 5XFAD AD-Tg mice, and if so, whether this would affect systemic Treg cells and CC activation for trafficking mo-MF. We therefore subjected 5XFAD AD-Tg mice to a weekly GA regimen for 4 weeks (hereinafter referred to as “weekly GA”; schematically depicted in Fig. 8A). We found that 5XFAD AD-Tg mice treated with weekly GA showed reduced neuroinflammation (Figure 8B-D) and improved cognitive performance that lasted up to 2 months post-treatment (Figure 8E-I). By flow cytometry studying the effects of weekly GA on systemic immunity and on the CC, we found a decrease in the levels of Foxp3 ⁺ Treg splenocytes (Fig. 9A) and an increase in IFN-γ producing cells in the CC of treated 5XFAD AD-Tg mice, with levels reaching , similar to those observed in wild-type controls (Fig. 9B). Increased levels of IFN-γ-expressing cells in the CC of mice treated with weekly GA were accompanied by up-regulation of epithelial expression of leukocyte trafficking molecules ( Fig. 9C ).

[165] To detect entry of penetrative mo-MF into the CNS, we used 5XFAD AD-Tg/CX ₃ CR1 ^GFP/+ mice with bone marrow chimeras (prepared using a head guard), which allowed visualization of circulating (green fluorescent protein-labeled ( GFB/GFP) ⁺ ) myeloid cells (Shechter et al, 2009; Shechter et al, 2013). We found increased homing of GFP ⁺ mo-MF into the CC and adjacent ventricular spaces following weekly GA therapy compared to guidewire-treated AD-Tg/CX ₃ CR1 ^GFP/+ controls (Fig. 9D-E). Immunohistochemistry of brain parenchyma showed the presence of GFP ⁺ mo-MF accumulations at sites of brain plaque formation (Fig. 9F), and quantification of infiltrating myeloid cells by flow cytometry analysis of the hippocampus in non-chimeric AD-Tg mice showed an increase in the number of CD11b ^high CD45 ^high -expressing cells (Fig. 9G,H). Together, these results support a functional link between mo-MF recruitment to sites of AD pathology, decreased systemic Treg levels, and IFN-γ-dependent CC activation.

Example 4. Interference with the activity of regulatory T cells using a small molecule inhibitor of histone acetyltransferase.

[166] The above data suggesting that Treg-mediated systemic immunosuppression interferes with the ability to fight AD pathology are reminiscent of the function attributed to regulatory T cells in cancer immunotherapy, in which these cells interfere with the ability of the immune system to mount an effective anti-tumor response (Bos & Rudensky, 2012; Nishikawa & Sakaguchi, 2010). We therefore concluded that treatments that directly interfere with Foxp3 ⁺ Treg cell activity may be effective in AD. We tested p300i (C646 (Bowers et al, 2010)), a non-peptide inhibitor of p300, a histone acetyltransferase that regulates Treg function (Liu et al, 2013); this inhibitor has been demonstrated to affect Treg suppressive activity, leaving protective T effector cell responses unaffected (Liu et al, 2013). We found that p300i-treated mice, compared with vehicle (DMSO)-treated mice, exhibited increased levels of systemic IFN-γ-expressing cells in the spleen (Fig. 10A) as well as in the CP (Fig. 10B). We then treated AD-Tg mice with either p300i or vehicle for 1 week, and examined the animals 3 weeks later for cerebral Aβ plaque load. Immunohistochemical analysis showed a significant reduction in cerebral Aβ plaque burden in p300i-treated mice compared to AD-Tg mice (Fig. 10C-E). We also tested whether the effects on plaque pathology after one course of treatment would last longer than 3 weeks, and if so, whether additional courses of treatment would contribute to the long-term effects. For this reason, we compared AD-Tg mice that received one course of p300i treatment and were examined 2 months later with an age-matched group that received two courses of treatment during this period with a 1-month interval between them (schematically depicted in Fig. 10F). We found that the reduction in cerebral plaque burden was evident even two months after one course of treatment, but was greater in mice that received two courses of treatment with a 1 month interval between them (Fig. 10G). Because impaired synaptic plasticity and memory in AD are associated with increased levels of soluble Aβ _1-40 /Aβ _1-42 (sAβ) in the brain (Shankar et al, 2008), we also measured sAβ levels after one or more cycles of p300i treatment. Again, we found that both one and two courses (1 month apart) were effective in reducing cerebral sAβ, but the effect was greater after multiple courses on sAβ _1-42 (Figure 10H). . These results indicated that although a single short course of treatment was effective, a series of treatments would maintain long-term therapeutic effects similar to our observations after weekly GA treatment.

Example 5: Therapeutic potential of PD-1 immune checkpoint blockade in Alzheimer's disease.

[167] We first tested whether targeting the PD-1 inhibitory pathway could affect IFN-γ-associated systemic immunity in AD-transgenic (AD-Tg) 5XFAD mice that coexpress five mutations associated with familial AD (Oakley et al , 2006). AD-Tg mice at 10 months of age, when cerebral pathology develops, were given both blocking antibodies directed against control PD-1 antibodies (anti-PD-1) and IgG by two intraperitoneal injections on days 1 and 4, and then on 7th day. Flow cytometry analysis showed that blockade of the PD-1 pathway resulted in increased frequencies of IFN-γ-producing CD4 ⁺ T splenocytes (Fig. 11A, B).

[168] We next examined whether this systemic immune response influences SS activity. Genomic RNA sequencing of CC (not shown; full analysis will be disclosed in the inventors' report entitled Example 5, and is available from the inventors upon request) showed an expression profile associated with response to IFN-γ (Fig. 11D and Table 3) , and real-time quantitative PCR (RT-qPCR) analysis confirmed increased IFN-γ mRNA levels in CCs compared with IgG-treated or untreated AD-Tg controls (Figure 11C). These findings confirmed a systemic and CC tissue-specific IFN-γ immune response following PD-1 blockade and prompted us to subsequently test the impact on disease pathology.

[169] To study the functional consequences of PD-1 blockade on AD pathology, we treated 10-month-old AD-Tg mice with anti-PD-1 or control IgG antibodies and assessed the effects on spatial learning and memory using the radial arm water maze (RSWM).

[170] One month after treatment (two intraperinetoneal injections 3 days apart), AD-Tg mice treated with anti-PD1 showed significant improvement in cognitive function compared to age-matched IgG-treated or untreated controls, achieving cognitive levels similar to those of age-matched wild-type mice (Figure 12A). We next examined whether the effects of PD-1 blockade on cognition in AD-Tg mice would last beyond 1 month and whether additional treatment sessions would be effective. We treated AD-Tg mice at 10 months of age with anti-PD-1 (“1 session”) or at 10 and 11 months of age (“2 sessions”), and examined cognitive outcomes at 12 months of age (schematically depicted in Fig. .12V). Control groups included wild-type mice, untreated AD-Tg mice, and AD-Tg mice that received two sessions of IgG treatment. We found that while a single session of anti-PD-1 administration had beneficial effects on spatial learning and memory 1 month after treatment (Figure 12A), no significant effect could be detected in mice that received a single session of treatment and were examined after 2 months (Fig. 12B). In contrast, AD-Tg mice that received two sessions of anti-PD-1 treatment, 1 month apart, exhibited cognitive performance similar to that of wild-type mice at the end of the 2-month period (Fig. 12B).

[171] We examined whether PD-1 blockade affected AD pathology manifested by cerebral Aβ plaque burden and gliosis. The brains of AD-Tg mice that received anti-PD-1 or IgG for one or two sessions were examined by immunohistochemistry for Aβ and glial fibrillary acidic protein (GFAP). We found that cerebral Aβ plaque burden was reduced in the dentate gyrus of the hippocampus (Figure 13A, B) as well as in the cerebral cortex (layer 5) (Figure 13A, C), two brain regions showing consistent pathology Aβ plaques in 5XFAD mice (Oakley et al, 2006). The effect on Aβ clearance appeared after one session of anti-PD-1 administration and became more durable after two sessions. Quantitative analysis of GFKB immunostaining showed reduced hippocampal astrogliosis in both 1-session and 2-session PD-1 blockade AD-Tg mice relative to IgG-treated controls (FIG. 13A,D). To investigate the effect of dosing and frequency of administration, female 5XFAD AD transgenic mice (mean population age 6 months) were treated with either an anti-PD-1-specific antibody (anti-mouse PD-1 IgG2a or an IgG control (rat IgG2a). treated with anti-PD-1, received either one injection of 500 μg antibody on day 1 of the experiment, or two injections of 250 μg with a 3-day interval between injections. Age-matched wild-type mice were used as an additional control group. Treatment effect spatial learning and memory performance of 5XFAD mice treated with anti-PD-1 - one injection (n=7) or two injections (n=11), IgG2a-treated 5XFAD (n=10) and WT (n=14) mice were assessed using the radial arm water maze (RAWM) at 7 months of age (Figure 14).Black arrows indicate treatment time points, illustrations indicate cognitive testing time points.Repeated measures were analyzed using a two-way ANOVA test and Dunnett's post hoc test. The magnitude of the error represents the mean ± standard error of the mean; *P<0.05, **P<0.01, ***P<0.001, anti-PD-1 treated (1 injection) versus IgG treated controls. One month after treatment (two intraperinetoneal injections 3 days apart), AD-Tg mice treated with anti-PD1 showed significant improvement in cognitive function compared to age-matched IgG-treated or untreated controls, achieving cognitive levels similar to levels in age-matched wild-type mice (Fig. 14).

[172] Finally, male 5XFAD AD transgenic mice were treated with repeated sessions, once a month, with either anti-PD-1-specific antibodies (IgG2a anti-mouse PD-1) or an IgG control (rat IgG2a). The first injection was given at the age of 3 months, the second at the age of 4 months, and the third at the age of 5 months. The dosage is indicated in the experimental design (Fig. 15A). Age-matched wild-type mice were used as an additional control group. The effect on spatial learning and memory was assessed using the radial arm water maze (RAWM) at two different time points - 5 months of age (Fig. 15B) and 6 months of age (Fig. 15C). Black arrows indicate treatment time points, illustrations show cognitive testing time points. Results in RRVL in 5XFAD mice treated with anti-PD-1 (n=7), 5XFAD mice treated with IgG2a (n=9) and wild-type controls (n=8). Repeated measures were analyzed using two-way ANOVA and Dunnett's post hoc test. The magnitude of the error represents the mean ± standard error of the mean; *P<0.05, **P<0.01, ***P<0.001, anti-PD-1 treated versus IgG treated controls. At 5 months of age, IgG-treated control mice had not completely lost spatial learning/memory skills and thus showed some learning during the final test on day 2 (Figure 15B), whereas at 6 months of age, disease progression was observed. with a subsequent decrease in functional results (Fig. 15C, D). Fig. 15D illustrates the decline in IgG-treated mice between 5 and 6 months of age, while the anti-PD-1 group retained learning ability. These data suggest that repeated treatments with PD-1 blockade can not only reverse disease progression when 5XFAD is administered to mice late in the disease, but also delay disease onset if treatment is started early in life, prior to cognitive decline (Fig. 15B-D).

[173] In AD, neuronal loss and synaptic failure were reported to be most strongly correlated with decline in spatial learning/memory skills. 5XFAD transgenic mice are one of the few animal models of AD that exhibit significant neuronal loss, which becomes evident in these mice at 6 months of age. We therefore assessed neuronal survival in mice following the experiment described above (Fig. 15A-D) after the last behavioral test. We analyzed neuronal survival in the brains of these mice, focusing on the subiculum, previously used to demonstrate neuronal loss in this mouse model of AD. Immunohistochemical analysis revealed a higher number of Neu-N ⁺ cells in the subiculum of 5XFAD mice treated with anti-PD-1 antibody compared to the IgG-treated group (Figure 15E,F).

[174] The molecular mechanisms underlying neuronal loss in AD have been linked to the activation of neuronal caspase-3. We therefore assessed whether neuronal rescue after anti-PD-1 treatment was associated with decreased levels of activated caspase-3 in Neu-N ⁺ cells. We found that mice treated with anti-PD-1 showed decreased activated caspase-3 immunoreactivity in Neu-N ⁺ neurons compared to the IgG-treated group (Figure 15G,H), further supporting the effect of anti-PD treatment -1 on neuron survival.

Example 6: Therapeutic potential of TIM-3 immune checkpoint blockade in Alzheimer's disease.

[175] To investigate the functional impact of TIM-3 blockade on AD pathology, we treated 6-month-old female 5XFAD AD-Tg mice with either an anti-TIM-3-specific antibody (TIM-3 anti-mouse) or an IgG control (IgG2a antibody rats). The dosage is indicated in the experimental design (Fig. 16). Treatment consisted of two intraperitoneal injections of antibody, 250 μg each, with a 3-day interval between injections. Age-matched wild-type mice were used as an additional control group. The effect of treatment on spatial learning and memory performance of anti-TIM-3-treated 5XFAD mice (n=9), IgG-treated 5XFAD mice (n=6), and wild-type controls (n=7) was assessed using a radial sleeve water maze (WRWL) at the age of 7 months (Fig. 16). Black arrows indicate treatment time points, illustrations show cognitive testing time points. Repeated measures were analyzed using two-way ANOVA and Dunnett's post hoc test. The magnitude of the error represents the mean ± standard error of the mean; *P<0.05, **P<0.01, ***P<0.001, anti-TIM-3 treated versus IgG treated controls. One month after treatment, AD-Tg mice treated with anti-TIM-3 showed significant improvement in cognitive performance compared to 5XFAD mice treated with IgG or age-matched wild-type controls (Fig. 16).

Example 7: Therapeutic potential of PDL1 immune checkpoint blockade in Alzheimer's disease and comparison with anti-PD-1 treatment.

[176] PD-1 is an inhibitory receptor expressed by numerous immune cells, including effector CD4 T cells, while its ligand PDL1 is expressed by dendritic cells, epithelial cells, and regulatory T cells. This raised the question of whether blocking PDL1 could have an effect similar to blocking PD-1 (Fig. 17A). To this end, 6-month-old 5XFAD mice were first administered a single dose of anti-PDL1 antibody (0.1 mg, 0.5 mg, or 1.5 mg per mouse) via intraperitoneal injection. Mice were examined 1 month later, at 7 months of age, using RRVL. While 0.1 mg anti-PDL1 had no effect on cognitive performance compared with IgG isotype control treatment, both doses (0.5 mg and 1.5 mg) had similar beneficial effects on task performance in the RRVL. (Fig. 17B). We then compared the effect of a single injection of 0.5 mg anti-PD-1 antibody and 0.5 mg anti-PDL1 antibody on cognitive performance, and therefore on brain pathology (Fig. 17C-J). Using the RRBL experiment, we found that treatment with anti-PDL1 antibody was as effective as anti-PD-1 antibody in improving functional cognitive outcomes in 5XFAD mice (Figure 17C). Immunohistochemical analysis of the brains of these mice after behavioral tests showed similar reductions in plaque burden (measured by plaque number and area covered by plaques) in the hippocampus (HC) and cortex, and in gliosis (measured by glial fibrillary acidic protein (GFAP) immunoreactivity) as in the group , treated with anti-PD-1 and anti-PDL1 group, compared with the IgG group (Fig. 17D). In the brains of mice treated with either anti-PD-1 or anti-PDL1 antibody, we also observed higher levels of immunoreactivity consistent with synaptophysin, a marker of presynaptic activity, indicating better synaptic preservation (Figure 17H, I). We analyzed il-12p40 and il-10 levels in the hippocampus of treated 5XFAD mice by quantitative real-time PCR. The results showed a shift towards anti-inflammatory activity in the hippocampus of mice treated with either anti-PD-1 or anti-PDL1, as evidenced by a decrease in the il-12p40/il-10 ratio compared to 5XFAD animals treated with IgG (Fig. 17J). It should be noted that the isotype-matched control antibodies for anti-PD-1 and anti-PDL1 are IgG2a and IgG2b, respectively. However, they showed similar results in all of our behavioral studies, so these two control groups were combined in the final analyzes presented here.

[177] PDL1 is expressed by activated immune cells such as T cells, B cells, macrophages, dendritic cells and microglia, as well as non-immune cells such as endothelial and epithelial cells. Thus, we hypothesized that expression of PDL1 by CC epithelium may contribute to the negative regulation (down-regulation) of leukocyte trafficking into the CNS by silencing the activity of IFN-γ-producing T cells that express PD-1 when communicating with PDL1-expressing epithelial cells within the CC . Immunohistochemical analysis shows that in adult mice, the CC epithelium expressed significantly higher levels of PDL1 compared to young mice (Fig. 18).

Example 8: Therapeutic potential of immune checkpoint blockade in Alzheimer's disease.

[178] To test whether immune checkpoint blockade can attenuate AD pathology, AD-Tg mice are treated at 6 to 10 months of age with one of the following anti-checkpoint antibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L, anti-CD80, anti-CD86, anti-B7-H3, anti-B7-H4, B7-H7, anti-BTLA, anti-HVEM, anti-CD137, anti-CD137L, anti- OX40L, anti-CD-27, anti-CD70, anti-STING, anti-TIGIT or anti-GITR. Some mice are treated with anti-PD-1 antibody as a positive control, IgG as a negative control, or a combination of anti-PD1 and one of the other checkpoint antibodies mentioned above. Treatment effects on spatial learning and memory are measured one month after treatment using the radial arm water maze (RSWM), Aβ plaque burden using Aβ immunohistochemistry, and hippocampal astrogliosis using glial fibrillary acidic protein (GFAP) immunohistochemistry.

[179] Antibody-treated mice are expected to show significant improvement in cognitive performance compared to IgG-treated and untreated AD-Tg mice, as well as a significant reduction in cerebral plaque burden.

Example 9: Therapeutic potential of PD-1 blockade in combination with the CTLA-4 immune checkpoint in Alzheimer's disease.

[180] At 10 months of age, 5XFAD transgenic (Tg) mice with Alzheimer's disease (AD) are administered intraperitoneally with either 250 μg anti-PD1 (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.) and 250 μg anti-CTLA. -4 (InVivoMAb anti-mCD152; #BE0131; Bioxcell Lifesciences Pvt. LTD.), or IgG control (IgG2a, #BE0089 or Polyclonal Syrian Hamster IgG, #BE0087; Bioxcell Lifesciences Pvt. LTD.) antibodies, on the 1st and day 4 of the experiment, and then examined 3 weeks later for cognitive performance using spatial learning and memory tasks in the radial arm water maze (RAWM) as described above.

[181] Some mice undergo an additional session with a 3-week treatment break. Control groups either receive IgG or no treatment, and all groups of mice are examined for cognitive performance after 3 weeks.

[182] Mice treated with the antibody combination are expected to show significant improvements in cognitive performance compared to IgG-treated and untreated AD-Tg mice, as well as a significant reduction in cerebral plaque burden.

Example 10. Therapeutic potential of immune checkpoint blockade in PTSD pathology.

[183] Particularly stressful conditions or chronic stress can lead to depression and post-traumatic stress disorder (PTSD). We adopted a PTSD-like physiological animal model in which mice exhibit insomnia, attention deficits, increased risk taking, and poor sleep (Lebow et al, 2012). In this experimental model of PTSD induction, mice are habituated over 10 days to a reverse day/night cycle, exposed to two electric shocks (trauma and trigger), called “PTSD induction,” and assessed at different time points post-injury. After a traumatic event, mice are injected with the drug, which blocks immune checkpoints. Mice are treated with one of the following regimens:

[184] Mice are treated with one of the following checkpoint antibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L anti-CD80, anti-CD86, anti-B7-H3, anti-B7- H4, B7-H7, anti-BTLA, HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70, anti-STING, anti-GITR or anti-TIGIT alone or in combination with anti-CTLA-4 antibody. Some mice are treated with anti-PD-1 antibody as a positive control, IgG as a negative control, or a combination of anti-PD1 and one of the other checkpoint antibodies mentioned above.

[185] Some mice receive an additional treatment session after an appropriate interval.

[186] Treated mice are not expected to exhibit anxiety-like behavior associated with PTSD in this experimental model, as assessed by time spent exploring and assessing risk in the dark/light maze or performing other behavioral tasks described in (Lebow et al, 2012).

Example 11. Therapeutic potential of the immune checkpoint blockade method in the pathology of Parkinson's disease.

[187] This experiment uses transgenic (Tg) mice with Parkinson's disease (PD) or MPTP-induced PD mouse models. Mice are treated in advanced stages of the disease using one of the following regimens:

[188] PD-Tg mice are treated with one of the following anti-checkpoint antibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L, anti-CD80, anti-CD86, anti-B7-H3, anti-B7-H4, B7-H7, anti-BTLA, HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70, anti-STING, anti-GITR or anti-TIGIT alone or in combination with anti-CTLA-4 antibody. Some mice are treated with anti-PD-1 antibody as a positive control, IgG as a negative control, or a combination of anti-PD1 and one of the other checkpoint antibodies mentioned above.

[189] Motor neurological functions are assessed, for example, using the rotarod test, which assesses the ability of mice to stay on a rotating drum.

[190] PD-Tg mice treated with one treatment are expected to exhibit significantly improved locomotor activity compared to the IgG or guide-treated control group or the untreated group. PD-Tg mice that receive two treatment sessions and are studied after an appropriate break are expected to exhibit long-term therapeutic effects. To maintain the therapeutic effect, mice are subjected to an active treatment session with an appropriate no-treatment break after each treatment session.

Example 12: Therapeutic potential of PD-1 in combination with CTLA-4 immune checkpoint blockade in Huntington's disease.

[191] The model used in these experiments may be a test system for Huntington's disease (HD) in R6/2 transgenic (Tg) mice. R6/2 transgenic mice overexpress mutated human huntingtin, which implies the introduction of multiple CAG repeats in mice at advanced stages of the disease. These mice exhibit progressive behavioral-motor deficits, beginning as early as 5-6 weeks of age and leading to premature death after 10-13 weeks. Symptoms include low body weight, muscle contractions, tremors and seizures.

[192] Mice are treated with one of the following regimens at 45 days of age:

[193] Mice are treated with one of the following checkpoint antibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L, anti-CD80, anti-CD86, anti-B7-H3, anti-B7 -H4, B7-H7, anti-BTLA, HVEM, anti-CD137, anti-CD137L, anti-OX40L, anti-CD-27, anti-CD70, anti-STING, anti-GITR or anti-TIGIT alone or in combination with anti-CTLA-4 antibody. Some mice are treated with anti-PD-1 antibody as a positive control, IgG as a negative control, or a combination of anti-PD1 and one of the other checkpoint antibodies mentioned above.

[194] Motor neurological functions are assessed, for example, using the rotarod test, which assesses the ability of mice to stay on a rotating drum.

[195] HD-Tg mice treated with one treatment are expected to exhibit significantly improved locomotor activity compared to the IgG or guide-treated control group or the untreated group. HD-Tg mice that undergo two treatment sessions and are studied after an appropriate break are expected to exhibit long-term therapeutic effects. To maintain the therapeutic effect, mice are subjected to an active treatment session with an appropriate no-treatment break after each treatment session.

Example 13. Therapeutic potential of the immune checkpoint blockade method in the pathology of amyotrophic lateral sclerosis.

[196] The model used in this experiment may represent transgenic mice overexpressing the defective human mutant allele of SOD1 containing the Gly93→Ala (G93A) gene (B6SJL-TgN (SOD1-G93A)1Gur (hereinafter “ALS mice”). In this model develops motor neuron disease, thus it is an acceptable model for testing ALS.

[197] Mice are treated with one of the following regimens at 75 days of age:

[198] Mice are treated with one of the following checkpoint antibodies: anti-ICOS, anti-B7RP1, anti-VISTA, anti-CD40, anti-CD40L, anti-CD80, anti-CD86, anti-B7-H3, anti-B7 -H4, B7-H7, anti-BTLA, HVEM, anti-CD137, anti-CD137L anti-OX40L anti-CD-27, anti-CD70, anti-STING, anti-GITR or anti-TIGIT alone or in combination with anti-CTLA-4 antibody. Some mice are treated with anti-PD-1 antibody as a positive control, IgG as a negative control, or a combination of anti-PD1 and one of the other checkpoint antibodies mentioned above.

[199] Motor neurological function is assessed, for example, using the rotarod test, which assesses the ability of mice to remain on a rotating drum, or where mice are allowed to grasp and hold onto a vertical wire (2 mm diameter) with a small loop at the lower end. The vertical wire allows mice to use both forelimbs and lower limbs for gripping. The wire in a vertical position makes circular movements (circle radius 10 cm) at a speed of 24 rpm. The time during which the mouse can hang on the wire is counted by a timer.

[200] ALS mice treated with one treatment are expected to exhibit significantly improved locomotor activity compared to the IgG or guide-treated control group or the untreated group. ALS mice that receive two treatment sessions and are studied after an appropriate break are expected to exhibit long-term therapeutic effects. To maintain the therapeutic effect, mice are subjected to an active treatment session with an appropriate no-treatment break after each treatment session.

Example 14 Dose-response experiments to determine the minimum and maximum dose range and experiments to determine the treatment regimen and its long-term therapeutic effect.

[201] We have already shown that a single treatment session using PD-1 blockade results in a significant reduction in plaque burden and improved cognitive function, lasting at least 2 months after treatment until the last time point tested. Here we describe a dose-response study using two additional doses administered to 5XFAD AD transgenic mice. The information sample will include amyloid plaque burden at one, two and three months after administration. Study groups will consist of 1) untreated 5XFAD mice; 2) 5XFAD mice receiving 1 injection of 500 μg control anti-PD-1 (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.); 3) 5XFAD mice receiving 1 injection of 250 μg control anti-PD-1 (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.); 4) 5XFAD mice receiving 1 injection of 100 μg control anti-PD-1 (RMP1-14; #BE0146; Bioxcell Lifesciences Pvt. LTD.); and 5) 5XFAD mice receiving 1 injection of 500 μg control anti-PD-1 (IgG2a; #BE0089; BioxcellLifesciencesPvt. LTD.). All mice were treated at the beginning of the experiment, and mice from each group were sacrificed and their brains examined at intervals of 1 month, 2 months, and 3 months after the start of treatment.

[202] Mice treated with anti-PD-1 antibodies are expected to show a significant reduction in beta-amyloid plaque burden in the brain compared to untreated AD-Tg mice or mice treated with an IgG control.

[203] An additional session of anti-PD-1 treatment one month after the initial treatment was found to maintain the effect of improving cognitive performance in 5XFAD AD-Tg mice (Example 5). These data suggest that repeated treatment sessions are necessary for long-term effectiveness. Here we describe a study that uses repeated injections to maintain long-term treatment effects.

[204] 5XFAD AD-Tg mice are injected with the drug at a dosage that will be determined based on the results of the previous study. The mice will be injected and their cognitive performance will be monitored using a radial arm water maze task during and after the study. Histological examination of the brain is also performed to determine the burden of amyloid plaques.

[205] Different groups of mice were given multiple doses of single injections (or double injections 3 days apart as described in Example 5) with no-treatment intervals of 2, 3, or 4 weeks (Table 4). Mice are monitored as described above one, two, or three months after initial treatment.

Example 15: Systemic administration of anti-PD-1 monoclonal antibody in RCS rats attenuates retinal degeneration.

[206] The purpose of this experiment was to determine whether systemic administration of an anti-PD1 antibody attenuates outer nuclear layer degeneration in an animal model of retinal degeneration.

[207] RCS rats, an established animal model of dry age-related macular degeneration, retinitis pigmentosa, and other degenerative diseases and conditions of the retina, contain a deletion mutation in the gene encoding the MerTK protein, which results in retinal degeneration and complete loss of vision by three months of age. A significant and rapid decrease in the thickness of the outer nuclear layer of the retina (ONL) is observed at 4 weeks of age. Thus, it is believed in the scientific literature that there is a direct correlation between preservation of ONL thickness in this model and preservation of vision.

[208] RCS rats were administered intraperitoneally at 4 weeks of age with either anti-PD1 monoclonal antibodies (total 760 μg per animal, n=10) or a matched IgG control (IgG2a) at the same concentration (n=10). Another group of RCS rats remained untreated (n=4). Two weeks after treatment, at 6 weeks of age, animals were sacrificed, their eyes were excised, and the thickness of the outer nuclear layer of the retina in each eye was determined by histological analysis using hematoxylin and eosin staining (H&E stain). Treatment effectiveness was determined by measuring ONL thickness along the entire length of the retina and plotting the data horizontally to create a map that allows treatment results to be determined in any region of the retina.

[209] Analysis of the average ONL thickness of all treated animals, with each individual eye serving as an independent data set, showed that at 6 weeks of age (2 weeks after treatment), the ONL in the central region of the retina in close proximity to the optic nerve head was significantly thicker in anti-PD1-treated animals compared to IgG-treated and untreated controls (Fig. 19A).

[210] To estimate effect sizes for treatment-responsive animals only, the threshold for classifying an animal as a “positive responder” was set at 2 standard error values above the mean central retinal thickness of the IgG control group. Based on this established cutoff value, 13 of 20 eyes analyzed (65%) in the anti-PD1 group were characterized as positive responders. In comparison, only 1 of 20 (5%) eyes analyzed were characterized as positive in the IgG experimental group; there is a significant difference (Chi-square = 15.82, P = 0.00007). When studying only 13 anti-PD1 positive eyes of this experimental group, it was shown that the thickness in the central region of the retina was 1.5-2 times greater than in control eyes. In addition, an even wider area of the central retina was significantly thicker than that of the control groups (Fig. 19B). Interestingly, although the antibody was administered systemically and was expected to reach both eyes and affect them at the same level, differences in treatment response between the two eyes of individual animals were observed in the four animals receiving anti-PD-1.

Example 16: Local administration of an anti-PD-1 monoclonal antibody directly into the vitreous in RCS rats attenuates retinal degeneration.

[211] The purpose of this experiment was to determine whether systemic administration of an anti-PD1 antibody attenuates outer nuclear layer degeneration in an animal model of retinal degeneration.

[212] RCS rats were treated at 4 weeks of age with either anti-PD1 monoclonal antibodies (n=6) or an appropriate IgG control (50 μg per animal). Injections were performed directly into the vitreous of one eye of each animal, while the contralateral eye was left without injection. Two weeks after treatment, at 6 weeks of age, animals were sacrificed, their eyes were excised, and the thickness of the outer nuclear layer of the retina in each eye was determined by histological analysis using hematoxylin and eosin staining (H&E stain). Treatment effectiveness was determined by measuring ONL thickness along the entire length of the retina and plotting the data horizontally to create a map that allows treatment results to be determined in any region of the retina.

[213] Analysis of mean ONL thickness in animals 2 weeks after treatment showed that in the anti-PD1 treatment group, the ONL was significantly thicker in several sections of the retina in the treated eye compared to the untreated contralateral eye (Figure 20A). This effect was not found in IgG-treated animals (Fig. 20B).

Example 17 Local blockade of PD-1 by intravitreal injections of PD-1 or PD-L1 monoclonal antibodies attenuates retinal degeneration.

[214] Anti-PD-L1 monoclonal antibodies or anti-PD-1 monoclonal antibodies, a combination of anti-PD-L1 and anti-PD-1 monoclonal antibodies, or suitable IgG fragments without the antigen-specific variable region will be injected directly into the vitreous body of aged RCS rats 4 weeks. Over the next 8 weeks, the animals' visual function will be assessed in response to visual stimuli. It is important to note that only one eye of each animal will receive treatment, while the contralateral eye will remain untreated and serve as an additional control. In addition, throughout the experiment, designated groups of RCS rats from each experimental group will be subjected to analysis aimed at quantitatively and qualitatively determining the effect of treatment on the retina in terms of neuronal survival and local immune response, as well as the effect on the retina of epithelial cells in terms of their expression of molecules trafficking of leukocytes and immune checkpoint ligands. Local blockade of the PD-1/PDL1 pathway via intravitreal injection is expected to result in attenuation of retinal degeneration, immune modulation, and preservation of visual function.

Example 18: Targeting the PD-1/PDL1 pathway in a mouse model of Tau pathology increases the recruitment of monocytic-derived macrophages to the brain parenchyma.

[215] Because immune checkpoint blockade treatment directly targets systemic immune cells, we hypothesized that its effectiveness would not be limited to the specific neuropathological feature associated with AD. To test whether treatments targeting the PD-1/PD-L1 pathway could be effective in other AD models that mimic distinct disease etiologies, we used a mouse model of AD that expresses two human-tau gene mutations (K257T/P301S; double mutant , DM-hTAU) associated with frontotemporal dementia. These mice develop neurofibrillary tangle (NFT)-predominant dementia, characteristic of a wide range of tauopathies, including Alzheimer's disease (AD) and other neurodegenerative diseases. Pathologies in these mice include cognitive deficits, neuroinflammation, glial cell activation, and phosphorylation of Tau proteins in the brain.

[216] We previously showed an inverse functional relationship between systemic immunosuppression and AD pathology in 5XFAD mice. Furthermore, in both mouse models of AD (5XFAD and J20), disease progression was found to be associated with loss of IFN-γ availability in the CC. This decrease in IFN-γ was accompanied by an increase in FoxP3 regulatory T cells in 5XFAD mice, and treatment with anti-PD-1 antibodies resulted in the restoration of IFN-γ signaling in the CC and the recruitment of monocytic-derived macrophages to the brain parenchyma (Baruch et al., 2016). Here, we first tested whether administration of an antibody directed against PD-L1 affected levels of systemic effector memory or regulatory T cells in DM-hTAU mice, as measured in the spleen 2 weeks after antibody administration. We found that also in this mouse model, in the absence of treatment, there is a systemic increase in FoxP3 regulatory T cells and a decrease in systemic levels of effector memory T cells (CD44 ⁺ CD-62L ^low ) compared to age-matched wild-type mice (Fig. 21A -WITH). Administration of anti-PDL1 antibody did not decrease the levels of suppressor cells but increased the level of effector memory T cells in IgG-treated mice (FIG. 21A-C) as analyzed by flow cytometry. We further analyzed the brains of the same mice to determine levels of monocyte-derived macrophages. We found a significant increase in the number of monocytic-derived macrophages in the brains of DM-hTAU mice treated with anti-PDL1 compared with those treated with the IgG isotype control (Figure 21D,E). These data support our hypothesis that this mouse model develops peripheral immunosuppression and that reducing immunosuppression in the periphery facilitates the entry of pathogenic leukocytes into the diseased brain. These results support our contention that immune checkpoint blockade may be applicable to AD characterized by additional etiologies.

Example 19: Blockade of the PD-1/PDL1 axis in a mouse model of Tau pathology mitigates cognitive deficits and cerebral pathology.

[217] Our finding that targeting systemic immune checkpoint pathways enhances monocyte trafficking into the brain parenchyma, as we observed in 5XFAD mice, prompted us to test the effect of targeting PD-1 or PD-L1 on cognitive function in a Tau mouse model . We treated DM-hTAU mice at 8 months of age with anti-PD-1 or anti-PDL1 using the same 0.5 mg dose as 5XFAD mice (Figure 22A); Isotype-matched antibodies directed against irrelevant antigens were used as negative controls (IgG-treated control). Age-matched wild-type mice were assessed as additional controls for normal learning/memory through the tests used in this study. In this mouse model of AD, short-term spatial memory is typically assessed using the T-maze and Y-maze tests. The two groups that received antibodies to PD-1 or PDL1 showed increased preference for the new arm in the T-maze test compared to the control group receiving IgG, 1 month after a single injection of antibodies (Fig. 22B, C). In addition, both experimental groups demonstrated improved cognitive performance in the Y-maze test compared to the IgG control group (FIG. 22D).

[218] The effect of treatment on gliosis revealed a decrease in GFKB immunoreactivity in the hippocampus of the anti-PD-1/PDL1 antibody-treated groups (Figure 22E, F). Next, we tested whether the beneficial effect of PD-1/PDL1 blockade on gliosis could be due to changes in the inflammatory cytokine milieu. To this end, we monitored the levels of various cytokines using real-time quantitative PCR (RT-qPCR) to determine whether the CNS inflammatory milieu was distorted by treatment. We found that both anti-PD-1 and anti-PDL1 reduced the expression levels of the pro-inflammatory cytokine, il-12p40, compared with the anti-inflammatory cytokine, il-10 in the hippocampus. In addition, both treatments resulted in decreased levels of the pro-inflammatory cytokines, tnfa and il-6 (Figure 22G).

[219] Neuroinflammation in animal models of Tau pathology has been shown to enhance hyperphosphorylation and disease progression. We therefore tested the effect of antibodies to PD-1 and PDL1 on Tau hyperphosphorylation. Immunohistochemical analysis of brain sections from DM-hTAU mice showed a decrease in immunoreactivity of AT-100 (Phospho-Tau Thr212, Ser214) and AT-180 (Phospho-Tau Thr231) epitopes in the CA1 and CA3 regions of the hippocampus, after blockade of anti-PD-1 and anti-PDL1 compared to the IgG isotype control group (FIG. 23A-F).

[220] Finally, since anti-PD-L1 antibody is used at a higher dose than anti-PD-1 antibody in cancer immunotherapy, we tested whether increasing the dose of anti-PDL1 antibody would have a superior effect. We therefore conducted an additional study testing the effects of different doses on short-term memory in both female and male (equally distributed among subgroups) DM-hTAU mice at 9 months of age. Mice were given a single injection of anti-PDL1 at 0.1, 0.5, or 1.5 mg per mouse, compared with a single injection of control antibody at 1.5 mg per mouse. Litter-matched wild-type mice were used as controls for intact cognitive abilities. Mice treated with 0.5 or 1.5 mg per mouse performed similar to wild-type mice; a dose of anti-PDL1 of 1.5 mg per mouse was slightly more effective, but the difference between doses was not significant (Fig. 23G).

[221] Taken together, these results indicate that systemic immune activation modifies key brain processes that are associated with disease pathology in an animal model of tau, similar to the pronounced effect found in animal models of beta-amyloid pathology.

Example 20: PD-1 blockade enhances hippocampal neurogenesis in 5XFAD mice.

[222] Doublecortin (DCX) is a microtubule-associated protein expressed by neuronal progenitor cells and immature neurons. In mature neuronal tissue, DCX is used as a marker of neurogenesis because it is expressed almost exclusively by developing neurons. Female 5XFAD mice (average population 6 months old) were treated with either PD-1 specific antibody (anti-mouse IgG2a PD-1; n=17) or IgG control (IgG2a rats; n=7) and sacrificed one month later. Age-matched wild-type mice were used as an additional control group. Parasagittal brain sections from representative animals were prepared, and the granular layer of the dentate gyrus was noted (Fig. 24A). Brain sections were immunized for the neuronal marker-NeuN (green), DCX (red), and Hoechst nuclear staining (blue). DCX+ cells were counted in a double-blind manner on 6-μm-thick brain sections (Figure 24B). Repeated measures were analyzed using one-way ANOVA test and Dunnett's post hoc test. The magnitude of the error represents the mean ± standard error of the mean; *P<0.05, **P<0.01, ***P<0.001. Results indicate that systemic blockade of the PD-1/PDL1 pathway induces protective immune activity that modulates the brain environment to support hippocampal neurogenesis, an impact on pathology that has been repeatedly correlated with beneficial effects on behavioral and cognitive deficits.

Example 21: PD-1 Blockade Increases Synaptic Plasticity in 5XFAD Mice.

[223] Vesicular glutamate transporter 1 (VGLUT1), expressed by glutamatergic neurons, mediates the uptake of glutamate into synaptic vesicles and has been shown to promote hippocampal synaptic plasticity and hippocampal-dependent spatial learning. Female 5XFAD mice (average population 6 months old) were treated with either PD-1 specific antibody (anti-mouse IgG2a PD-1; n=17) or IgG control (IgG2a rats; n=7) and sacrificed one month later. Age-matched wild-type mice were used as an additional control group. Parasagittal brain sections from representative animals were prepared and the subiculum area marked (Fig. 25A). Brain sections were immunostained for VgluT1. Fluorescence intensity was quantified in a double-blind manner using ImageJ software on 6-μm-thick brain sections (Figure 25B). Repeated measures were analyzed using one-way ANOVA test and Dunnett's post hoc test. The magnitude of the error represents the mean ± standard error of the mean; *P<0.05, **P<0.01, ***P<0.001. Results indicate that systemic blockade of the PD-1/PDL1 pathway induces protective immune activity in the periphery and modulates the brain environment to support synaptic plasticity and preserve cognitive function.

Example 22: PD-1 blockade reduces neuronal loss in the subiculum of 5XFAD mice.

[224] The 5XFAD transgenic mouse model is one of the few amyloid animal models that demonstrates significant neuronal loss similar to AD progression in humans. Neuronal loss in 5XFAD mice was characterized in the subiculum and cortical layer 5. Female 5XFAD mice (average cohort 6 months old) were treated with either a PD-1 specific antibody (anti-mouse PD-1 IgG2a; n=17) or an IgG control (IgG2a rats; n=7), they were sacrificed a month later. Age-matched wild-type mice were used as an additional control group. Parasagittal brain sections from representative animals were prepared and the subiculum area marked (Fig. 26A). Brain sections were immunostained for NeuN, highlighting neurons (in green).

Subiculum neurons were quantified in a double-blind manner using ImageJ software on 6-μm-thick brain sections (Figure 25B). Repeated measures were analyzed using one-way ANOVA test and Dunnett's post hoc test. The magnitude of the error represents the mean ± standard error of the mean; *P<0.05, **P<0.01, ***P<0.001. The results indicate that systemic blockade of the PD-1/PDL1 pathway induces protective immune activity that modulates the brain environment, making it permissive to support neuronal survival and rescue, ultimately promoting better cognitive performance.

[225] In conclusion, it should be understood that although aspects of the present specification are indicated by reference to specific embodiments, one skilled in the art will readily understand that the described embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter of the invention is in no way limited to any specific compound, composition, article, device, methodology, protocol and/or reagent, etc., described herein unless expressly stated therein. Moreover, those skilled in the art will appreciate that certain changes, modifications, permutations, alterations, additions, subtractions, and subcombinations thereof may be made to the subject matter in accordance with the principles described herein without departing from the spirit of this specification. It is therefore intended that the following appended claims and their claims set forth below shall be interpreted so as to include all such alterations, modifications, permutations, alterations, additions, subtractions and subcombinations as come within their true spirit and scope.

[226] Certain embodiments of the present invention are described herein, including the best mode of carrying out the invention known to the inventors. Of course, variations on these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventor expects that those skilled in the art will use such variations as needed, and the inventors anticipate that the present invention will be practiced differently than specifically described herein. Accordingly, this invention includes all modifications and equivalents to the subject matter set forth in the claims appended hereto to the extent permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible embodiments is also covered by the invention, unless otherwise indicated herein or unless the context clearly indicates otherwise.

[227] The grouping of alternative embodiments, elements, or steps of the present invention should not be construed as limiting. Each group member may be mentioned and stated separately or in any combination with other group members appearing herein. It is expected that one or more members of the group may be included in or removed from the group for reasons of convenience and/or patentability. Upon such inclusion or deletion, the specification is deemed to contain the modified group, thereby conforming to the written description of all Markush groups used in the appended claims.

[228] Unless otherwise indicated, all numbers expressing characteristic, element, quantity, parameter, property, period, etc., used in the present description and claims are to be understood as modified in all possible cases by the term “about.” As used herein, the term “about” means that the characteristic, element, quantity, parameter, property or period it defines includes a range of ten percent above or below the value of the specified characteristic, element, quantity, parameter, property or period. Accordingly, unless otherwise stated, figures set forth in the specification and appended claims are approximate and may vary. For example, since mass spectrometry instruments may vary slightly in determining the mass of a given analyte, the term "about" in the context of the mass of an ion or the ratio of an ion's mass to its charge means +/- 0.50 atomic mass units. Without intending to limit the application of the doctrine of equivalents to the scope of the claims, each numeric designation must, at a minimum, be construed in light of the number of significant figures attested and applying normal rounding techniques.

[229] The use of the terms “can” or “capable” in relation to an embodiment or aspect of an embodiment also carries the alternative meaning of “cannot” or “is not capable.” Thus, if the present specification states that an embodiment or aspect of an embodiment may be or is capable of being included as part of the subject matter of the invention, then a negative limitation or exclusionary condition is also expressly implied to mean that the embodiment or aspect of an embodiment may not be or are not capable of being included as part of the subject matter of the invention. Likewise, the use of the term “if required” in relation to an embodiment or aspect of an embodiment means that such embodiment or aspect of an embodiment may or may not be included as part of the subject matter. Whether such a negative limitation or exclusionary condition applies depends on whether the negative limitation or exclusionary condition is specified in the claimed subject matter.

[230] Although the numerical ranges and values establishing the broad scope of the invention are approximations, the numerical ranges and values stated in the specific examples are stated to the greatest possible accuracy. However, any numerical range or value inherently contains certain errors that inevitably arise as a result of the standard deviation in the associated test measurements. The indication of numeric value ranges here is intended only as a shorthand way of individually referring to each individual numeric value included in the range. Unless otherwise indicated, each individual numeric range value is included herein as if it were individually described herein.

[231] The terms “some,” “any,” “given,” and similar indications used in the context of the description of the present invention (especially in the context of the following claims) are to be construed to imply both the singular and the plural, unless otherwise stated. stated here or the context does not clearly contradict it. In addition, ordinal numbers such as "first", "second", "third", etc. are used with identified elements to distinguish them and do not indicate or imply the required or limited number of such elements, nor do they indicate the specific position or order of such elements unless expressly stated otherwise. All methods described here can be performed in any suitable order, unless otherwise noted here or the context clearly dictates. The use of any and all examples or phrases indicating examples (eg, “such as”) herein is intended only to better highlight the present invention and does not constitute a limitation on the scope of the invention otherwise claimed. Nothing in this specification should be construed as indicating any non-claimed element necessary to practice the invention.

[232] When used in a claim, whether as filed or as amended, the transitional non-limiting term “comprising” (and equivalent partial phrases such as including, encompassing and having) refers to all expressly identified elements , limitations, steps and/or features alone or in combination with unspecified objects of the invention; the named elements, limitations and/or features are important, but other not yet named elements, limitations and/or features may be added and still form a structure within the scope of the claims. Specific embodiments disclosed herein may be further limited in the claims by using the closed transition phrases “consisting of” or “consisting primarily of” instead of or as a modification of the term “comprising.” When used in a claim, whether as filed or as amended, the limiting phrase “consisting of” excludes any element, limitation, step or feature not expressly stated in the claim. The transitional limiting phrase "consisting primarily of" limits the scope of the claims to the expressly stated elements, limitations, steps and/or features and any other elements, limitations, steps and/or features that do not materially affect the essential and novel characteristics of the claimed subject matter. . Thus, the meaning of a transitional non-limiting phrase such as “comprising” is defined to include all of the specifically enumerated elements, limitations, steps and/or features, as well as any optional additional ones that have not been specified. The meaning of the transitive limiting phrase "consisting of" is defined to include only those elements, limitations, steps and/or features that are specifically set forth in the claims, while the meaning of the transitive limiting phrase "consisting primarily of" is defined to include only those elements, limitations , steps and/or features that are specifically indicated in the claims, and those elements, limitations, steps and/or features that do not significantly affect the basic and new characteristics of the claimed subject matter of the invention. Therefore, the transitional non-limiting phrase “comprising” (and equivalent transitional non-limiting phrases) includes in its limiting meaning the claimed subject matter of the invention indicated by the transitional limiting phrases “consisting of” or “consisting primarily of”. Thus, embodiments described herein or so stated by the phrase “comprising” are expressly or inherently clearly described, included, and substantiated herein with respect to the phrases “consisting primarily of” and “consisting of.”

[233] All patents, patent publications and other publications mentioned and identified herein are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methods described in such publications, which may be used in connection with the present invention. These publications are presented solely for their disclosure prior to the filing date of this application. Nothing in this regard shall be construed as an admission that inventors are not entitled to defer such disclosure by reason of prior invention or for any other reason. All statements of date or representation as to the contents of these documents are based on information available to the applicants and do not constitute an endorsement of the accuracy of the dates or contents of these documents.

[234] Finally, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to what is specifically presented and described.

REFERENCE DOCUMENTATION

• AkiyamaH, BargerS, BarnumS, BradtB, BauerJ, ColeGM, CooperNR, EikelenboomP, EmmerlingM, FiebichBL, FinchCE, FrautschyS, GriffinWS, HampelH, HullM, LandrethG, LueL, MrakR, MackenzieIR, McGeerPL, PachterJ, PasinettiG, Plata-SalamanC, RogersJ, RydeIR, ShenY, StreitW, StrohmeyerR, Tooyomal, VanMuiswinkelFL, VeerhuisR, WalkerD, WebsterS, WegrzyniakB, WenkG, Wyss-CorayT (2000) . Neurobiology of aging 21:383–421

• Alamed J, Wilcock D M, Diamond D M, Gordon M N, Morgan D (2006) Two-day radial-arm water maze learning and memory task; robust resolution of amyloid-related memory deficits in transgenic mice. Nature protocols 1:1671-1679

• Bai A, Lu N, Guo Y, Liu Z, Chen J, Peng Z (2009) All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. Journal of leukocyte biology 86:959-969

• BaruchK, DeczkowskaA, DavidE, CastellanoJM, MillerO, KertserA, BerkutzkiT, Barnett-ltzhakiZ, BezalelD, Wyss-CorayT, Amitl, SchwartzM (2014) Aging. Aging-induced type linterferon response at the choroid plexus negatively affects brain function. Science 346:89-93

• BaruchK, KertserA, PoratZ, SchwartzM (2015) CerebralnitricoxiderepresseschoroidplexusNFkappaB-dependentgatewayactivityforleukocytetrafficking. The EMBO journal

• BaruchK, Ron-HarelN, GalH, DeczkowskaA, ShifrutE, NdifonW, Mirlas-NeisbergN, CardonM, Vakninl, CahalonL, BerkutzkiT, MattsonMP, Gomez-PinillaF, FriedmanN, SchwartzM (2013) CNS-specificimmunityatthechoroidplexusshiftstowarddestructiveTh2 inflammationinbrainaging. Proceedings of the National Academy of Sciences of the United States of America 110:2264-2269

• Baruch, A. Deczkowska, N. Rosenzweig, A. Tsitsou-Kampeli, A. M. Sharif, O. Matkovitch-Natan, A. Kertser, E. David, I. Amit, M. Schwartz (2016) PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of disease, Nat. Med. 22, 135-137

• Borchelt D R, Ratovitski T, van Lare J, Lee M K, Gonzales V, Jenkins N A, Copeland N G, Price D L, Sisodia S S (1997) Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron 19:939–945

• Bos P D, Rudensky A Y (2012) Treg cells in cancer: a case of multiple personality disorder. Science translational medicine 4:164fs144

• BowersEM, YanG, MukherjeeC, OrryA, WangL, HolbertMA, CrumpNT, HazzalinCA, LiszczakG, YuanH, LaroccaC, SaldanhaSA, AbagyanR, SunY, MeyersDJ, MarmorsteinR, MahadevanLC, AlaniRM, ColePA (2010) Virtualligandscreeningofthep300/CBPhistoneacetyltransferase: identificationofaselectivesmall molecule inhibitor. Chemistry & biology 17:471-482

• Breitner J C, Haneuse S J, Walker R, Dublin S, Crane P K, Gray S L, Larson E B (2009) Risk of dementia and AD with prior exposure to NSAIDs in an elderly community-based cohort. Neurology 72:1899–1905

• Brestoff J R, Artis D (2013) Commensal bacteria at the interface of host metabolism and the immune system. Nature immunology 14:676-684

• Burgess A, Vigneron S, Brioudes E, Labbe J C, Lorca T, Castro A (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proceedings of the National Academy of Sciences of the United States of America 107:12564-12569

• Butovsky O, Koronyo-Hamaoui M, Kunis G, Ophir E, Landa G, Cohen H, Schwartz M (2006) Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like growth factor 1. Proceedings of the National Academy of Sciences of the United States of America 103:11784-11789

• Butovsky O, Kunis G, Koronyo-Hamaoui M, Schwartz M (2007) Selective ablation of bone marrow-derived dendritic cells increases amyloid plaques in a mouse Alzheimer's disease model. The European journal of neuroscience 26:413-416

• Chen X, Oppenheim J J (2011) Resolving the identity myth: key markers of functional CD4+FoxP3+ regulatory T cells. International immunopharmacology 11:1489-1496

• Colombo M P, Piconese S (2007) Regulatory-T-cell inhibition versus depletion: the right choice in cancer immunotherapy. Nature reviews Cancer 7:880–887

• Coyne G O, Gulley J L (2014) Adding fuel to the fire: Immunogenic intensification. Human vaccines & immunotherapeutics 10:3306-3312

• Dalotto-MorenoT, CrociDO, CerlianiJP, Martinez-AlloVC, Dergan-DylonS, Mendez-HuergoSP, StupirskiJC, MazalD, OsinagaE, ToscanoMA, SundbladV, RabinovichGA, SalatinoM (2013) Targeting galectin-1 overcomes breast cancer associated immunosuppression and prevents metastatic disease. Cancer research 73:1107-1117

• Duraiswamy J, Freeman G J, Coukos G (2014) Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumor response. Cancer research 74:633-634; discussion 635

• Francisco L M, Sage P T, Sharpe A H (2010) The PD-1 pathway in tolerance and autoimmunity. Immunological reviews 236:219-242

• Gabrilovich D I, Nagaraj S (2009) Myeloid-derived suppressor cells as regulators of the immune system. Nature reviews Immunology 9:162-174

• Galvin K S, Dyck L, Marshall N A, Stefanska A M, Walsh K P, Moran B, Higgins S C, Dungan L S, Mills K H (2013) Blocking retinoic acid receptor-alpha enhances the efficacy of a dendritic cell vaccine against tumors by suppressing the induction of regulatory T cells. Cancer immunology, immunotherapy: C// 62:1273-1282

• Ghiringhelli F, Bruchard M, Chalmin F, Rebe S (2012) Production of adenosine by ectonucleotidases: a key factor in tumor immunoescape. Journal of biomedicine & biotechnology 2012:473712

• Group A R, Lyketsos C G, Breitner J C, Green R C, Martin B K, Meinert C, Piantadosi S, Sabbagh M (2007) Naproxen and celecoxib do not prevent A D in early results from a randomized controlled trial. Neurology 68:1800–1808

• Hardy J, Selkoe D J (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297:353-356

• He F, Balling R (2013) The role of regulatory T cells in neurodegenerative diseases. Wiley interdisciplinary reviews Systems biology and medicine 5:153-180

• HirayamaM, NishikawaH, NagataY, TsujiT, KatoT, KageyamaS, UedaS, SugiyamaD, HoriS, SakaguchiS, RitterG, OldLJ, GnjaticS, ShikuH (2013) OvercomingregulatoryT-cellsuppressionbyalyophilizedpreparationofStreptococcuspyogenes. European journal of immunology 43:989-1000

• Hong J, Li N, Zhang X, Zheng B, Zhang J Z (2005) Induction of CD4+CD25+ regulatory T cells by copolymer-l through activation of transcription factor Foxp3. Proceedings of the National Academy of Sciences of the United States of America 102:6449-6454

• Joller N, Peters A, Anderson A C, Kuchroo V K (2012) Immune checkpoints in the central nervous system autoimmunity. Immunological reviews 248:122-139

• JuY, ShangX, LiuZ, ZhangJ, LiY, ShenY, LiuY, LiuC, LiuB, XuL, WangY, ZhangB, ZouJ (2014) The Tim-3/galectin-9 pathway involvesinthehomeostasisofhepaticTregsinamousemodelofconcanavalinA-inducedhepatitis. Molecular immunology 58:85-91

• Jung S, Aliberti J, Graemmel P, Sunshine M J, Kreutzberg G W, Sher A, Littman D R (2000) Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Molecular and cellular biology 20:4106-4114

• Kim J M, Rasmussen J P, Rudensky A Y (2007) Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nature immunology 8:191-197

• KimPS, JochemsC, Grengal, DonahueRN, TsangKY, GulleyJL, SchlomJ, FarsaciB (2014) Pan-Bcl-2 inhibitor, GX15-070 (obatoclax), decreaseshumanTregulatorylymphocyteswhilepreservingeffectorTlymphocytes: arationaleforitsuseincombinationimmunotherapy. Journal of immunology 192:2622-2633

• Kotsakis A, Harasymczuk M, Schilling B, Georgoulias V, Argiris A, Whiteside T L (2012) Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples. Journal of immunological methods 381:14-22

• Kunis G, Baruch K, Miller O, Schwartz M (2015) Immunization with a Myelin-Derived Antigen Activates the Choroid Plexus for Recruitment of Immunoregulatory Cells to the CNS and Attenuates Disease Progression in a Mouse Model of ALS. The Journal of neuroscience: the official journal of the Society for Neuroscience 35:6381-6393

• Kunis G, Baruch K, Rosenzweig N, Kertser A, Miller O, Berkutzki T, Schwartz M (2013) IFN-gamma-dependent activation of the choroid plexus for CNS immune surveillance and repair. Brain: a journal of neurology 136:3427-3440

• Lebow M, Neufeld-Cohen A, Kuperman Y, Tsoory M, Gil S, Chen A (2012) Susceptibility to PTSD-like behavior is mediated by corticotropin-releasing factor receptor type 2 levels in the bed nucleus of the stria terminalis. J Neurosci 32:6906–6916

• Lesokhin A M, Callahan M K, Postow M A, Wolchok J D (2015) On being less tolerant: enhanced cancer immunosurveillance enabled by targeting checkpoints and agonists of T cell activation. Science translational medicine 7:280sr281

• LiuY, WangL, PredinaJ, HanR, BeierUH, WangLC, KapoorV, BhattiTR, AkimovaT, SinghalS, BrindlePK, ColePA, AlbeldaSM, HancockWW (2013) Inhibitionofp300impairsFoxp3(+)Tregulatorycellfunctionandpromotesantitumorimmunity. Nature medicine 19:1173-1177

• MarabelleA, KohrtH, Sagiv-Barfil, AjamiB, AxtellRC, ZhouG, RajapaksaR, GreenMR, TorchiaJ, BrodyJ, LuongR, RosenblumMD, SteinmanL, LevitskyHI, TseV, LevyR (2013) Depletingtumor-specificTregsatasinglesiteeradicatesdisseminatedtumors. The Journal of clinical investigation 123:2447-2463

• Mellman I, Coukos G, Dranoff G (2011) Cancer immunotherapy comes of age. Nature 480:480–489

• Michaud J P, Bellavance M A, Prefontaine P, Rivest S (2013) Real-time in vivo imaging reveals the ability of monocytes to clear vascular amyloid beta. Cell reports 5:646-653

• Nishikawa H, Sakaguchi S (2010) Regulatory T cells in tumor immunity. International journal of cancer Journal international cancer Ml: 759-767

• OakleyH, ColeSL, LoganS, MausE, ShaoP, CraftJ, Guillozet-BongaartsA, OhnoM, DisterhoftJ, VanEldikL, BerryR, VassarR (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic disease with five familial Alzheimer's disease mutations: potential factor sinamyloid plaque formation. The Journal of neuroscience: the official journal of the Society for Neuroscience 26: 10129-10140

• Ohaegbulam K S, Assal A, Lazar-Molnar E, Yao Y, Zang X (2015) Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends in molecular medicine 21:24-33

• Pardoll D M (2012) The blockade of immune checkpoints in cancer immunotherapy. Nature reviews Cancer 12:252–264

• PengW, LiuC, XuC, LouY, ChenJ, YangY, YagitaH, OverwijkWW, LizeeG, RadvanyiL, HwuP (2012) PD-1 blockadeenhancesT-cell migrationtotumorsbyelevatinglFN-gammainduciblechemokines. Cancer research 72:5209-5218

• PereH, MontierY, BayryJ, Quintin-ColonnaF, MerillonN, DransartE, BadoualC, GeyA, RavelP, MarcheteauE, BatteuxF, SandovalF, AdoteviO, ChiuC, GarciaS, TanchotC, LoneYC, FerreiraLC, NelsonBH, HanahanD, FridmanWH, JohannesL, TartourE (2011 ) ACCR4 antagonistcombinedwithvaccinesinducesantigen-specificCD8+ Tcellsandtumorimmunityagainstselfantigens. Blood 118:4853–4862

• Postow M A, Callahan M K, Wolchok J D (2015) Immune Checkpoint Blockade in Cancer Therapy. Journal of clinical oncology: official journal of the American Society of Clinical Oncology

• Qin A, Wen Z, Zhou Y, Li Y, Li Y, Luo J, Ren T, Xu L (2013) MicroRNA-126 regulates the induction and function of CD4(+) Foxp3(+) regulatory T cells through PI3K/ AKT pathway. Journal of cellular and molecular medicine 17:252-264

• Rosenkranz D, Weyer S, Tolosa E, Gaenslen A, Berg D, Leyhe T, Gasser T, Stoltze L (2007) Higher frequency of T regulatory cells in the elderly and increased suppressive activity in neurodegeneration. Journal of neuroimmunology 188:117-127

• RoyS, BarikS, BanerjeeS, BhuniyaA, PalS, BasuP, BiswasJ, GoswamiS, ChakrabortyT, BoseA, BaralR (2013) Neemleafglycoproteinovercomesindoleamine 2,3 dioxygenase-mediatedtoleranceindendriticcellsbyattenuatinghyperactiveregulatoryTcellsincervic alcancerstagelll Bpatients. Human immunology 74:1015–1023

• Sakaguchi S, Yamaguchi T, Nomura T, Ono M (2008) Regulatory T cells and immune tolerance. Cell 133:775–787

• SaresellaM, CalabreseE, Marventanol, PianconeF, GattiA, CalvoMG, NemniR, ClericiM (2010) PD1 negativeandPD1 positiveCD4+ Journal of disease JAD 21:927–938

• Schmidt S D, Nixon R A, Mathews P M (2005) ELISA method for measuring amyloid-beta levels. Methods in molecular biology 299:279-297

• Schreiber R D, Old L J, Smyth M J (2011) Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science 331:1565-1570

• Schwartz M, Baruch K (2014a) Breaking peripheral immune tolerance to CNS antigens in neurodegenerative diseases: boosting autoimmunity to fight-off chronic neuroinflammation. Journal of autoimmunity 54:8-14

• Schwartz M, Baruch K (2014b) The resolution of neuroinflammation in neurodegeneration: leukocyte recruitment via the choroid plexus. The EMBO journal 33:7-22

• ShankarGM, LiS, MehtaTH, Garcia-MunozA, ShepardsonNE, Smithl, BrettFM, FarrellMA, RowanMJ, LemereCA, ReganCM, WalshDM, SabatiniBL, SelkoeDJ (2008) Amyloid- Nat ure medicine 14:837-842

• Shechter R, London A, Varol C, Raposo C, Cusimano M, Yovel G, Rolls A, Mack M, Pluchino S, Martino G, Jung S, Schwartz M (2009) Infiltrating blood-derived macrophages are vital cells playing an anti -inflammatory role in recovery from spinal cord injury in mice. PLoS medicine 6: e1000113

• ShechterR, MillerO, YoveIG, RosenzweigN, LondonA, RuckhJ, KimKW, KleinE, KalchenkoV, BendelP, LiraSA, JungS, SchwartzM (2013) RecruitmentofbeneficialM2 macrophagestoinjuredspinalcordisorchestratedbyremotebrainchoroidplexus. Immunity 38:555–569

• Shevchenko I, Karakhanova S, Soltek S, Link J, Bayry J, Werner J, Umansky V, Bazhin A V (2013) Low-dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Pancreatic cancer model. International journal of cancer Journal international du cancer 133:98-107

• Simpson T R, Li F, Montalvo-Ortiz W, Sepulveda M A, Bergerhoff K, Arce F, Roddie C, Henry J Y, Yagita H, Wolchok J D, Peggs K S, Ravetch J V, Allison J P, Quezada S A (2013) Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. The Journal of experimental medicine 210:1695-1710

• Smith P M, Howitt M R, Panikov N, Michaud M, Gallini S A, Bohlooly Y M, Glickman J N, Garrett W S (2013) The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341:569-573

• Suffner J, Hochweller K, Kuhnle M C, Li X, Kroczek R A, Garbi N, Hammerling G J (2010) Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice. Journal of immunology 184:1810-1820

• Terme M, Colussi O, Marcheteau E, Tanchot C, Tartour E, Taieb J (2012) Modulation of immunity by antiangiogenic molecules in cancer. Clinical & developmental immunology 2012:492920

• Thomas-Schoemann A, Batteux F, Mongaret C, Nicco C, Chereau C, Annereau M, Dauphin A, Goldwasser F, Weill B, Lemare F, Alexandre J (2012) Arsenic trioxide exerts antitumor activity through regulatory T cell depletion mediated by oxidative stress in a murine model of colon cancer. Journal of immunology 189: 5171-5177

• Torres K C, Araujo Pereira P, Lima G S, Bozzi I C, Rezende V B, Bicalho M A, Moraes E N, Miranda D M, Romano-Silva M A (2013) Increased frequency of T cells expressing IL-10 in Alzheimer disease but not in late -onset depression patients. Progress in neuropsychopharmacology & biological psychiatry 47:40-45

• Vom Berg J, Prokop S, Miller K R, Obst J, Kalin R E, Lopategui-Cabezas I, Wegner A, Mair F, Schipke S G, Peters O, Winter Y, Becher B, Heppner F L (2012) Inhibition of IL- 12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline. Nature medicine 18:1812-1819

• Voo K S, Boyer L, Harline M L, Vien L T, Facchinetti V, Arima K, Kwak L W, Liu Y J (2013) Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function. Journal of immunology 191:3641-3650

• Walsh J T, Zheng J, Smirnov I, Lorenz U, Tung K, Kipnis J (2014) Regulatory T cells in central nervous system injury: a double-edged sword. Journal of immunology 193:5013-5022

• Ward F J, Dahal L N, Wijesekera S K, Abdul-Jawad S K, Kaewarpai T, Xu H, Vickers M A, Barker R N (2013) The soluble isoform of CTLA-4 as a regulator of T-cell responses. European journal of immunology 43:1274-1285

• Weber J S, Kudchadkar R R, Yu B, Gallenstein D, Horak S E, Inzunza H D, Zhao X, Martinez A J, Wang W, Gibney G, Kroeger J, Eysmans C, Sarnaik A A, Chen Y A (2013) Safety, efficacy, and biomarkers of nivolumab with vaccine in ipilimumab-refractory or -naive melanoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 31:4311-4318

• Weber M S, Hohlfeld R, Zamvil S S (2007) Mechanism of action of glatiramer acetate in the treatment of multiple sclerosis. Neurotherapeutics: the journal of the American Society for Experimental Neuro Therapeutics 4:647-653

• Weiskopf K, Ring A M, Schnorr P J, Volkmer J P, Volkmer A K, Weissman I L, Garcia K S (2013) Improving macrophage responses to therapeutic antibodies by molecular engineering of SIRPalpha variants. Oncoimmunology 2:e25773

• Wyss-Coray T, Rogers J (2012) Inflammation in Alzheimer disease-a brief review of the basic science and clinical literature. Cold Spring Harbor perspectives in medicine 2:a006346

• Zeng J, See A P, Phallen J, Jackson S M, Belcaid Z, Ruzevick J, Durham N, Meyer C, Harris T J, Albesiano E, Pradilla G, Ford E, Wong J, Hammers H J, Mathios D, Tyler B, Brem H, Tran P T, Pardoll D, Drake C G, Lim M (2013) Anti-PD-1 blockade and stereotactic radiation produce long-term survival in mice with intracranial gliomas. International journal of radiation oncology, biology, physics 86:343-349

• Zhao L, Sun L, Wang H, Ma H, Liu G, Zhao Y (2007) Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice. Journal of leukocyte biology 81:1386-1394

• Zheng H, Fridkin M, Youdim M (2015) New approaches to treating Alzheimer's disease. Perspectives in medicinal chemistry 7:1-8

Claims (5)

1. A method of treating tauopathy in an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising an anti-PD-1 antibody, an anti-PDL1 antibody or an anti-TIM-3 antibody, wherein the tauopathy is selected from Pick's disease, globular glial tauopathy, argyrophilic grain disease, chronic traumatic encephalopathy and primary age-related tauopathy (PART), formerly known as dementia with neurofibrillary tangles (NFT dementia), wherein the pharmaceutical composition is administered using a dosage regimen comprising at least two courses of treatment, each the course of treatment includes sequentially a treatment session in which the pharmaceutical composition is administered to the individual and a subsequent non-treatment session in which the pharmaceutical composition is not administered to the individual, the administration of the pharmaceutical composition during the treatment session is a single administration, and the non-treatment session is from one month to three months. 2. The method of claim 1, wherein the anti-PD-1 antibody is a neutralizing anti-PD-1 antibody, the anti-PD-L1 antibody is a neutralizing anti-PDL1 antibody, or an anti-TIM-3 antibody. is a neutralizing anti-TIM-3 antibody. 3. The method of claim 2, wherein the anti-PD-1 antibody is a neutralizing human anti-PD-1 antibody or a humanized neutralizing anti-PD-1 antibody. 4. The method of claim 2, wherein the anti-PD-L1 antibody is a neutralizing human anti-PD-L1 antibody or a humanized neutralizing anti-PD-L1 antibody. 5. The method of claim 2, wherein the anti-TIM-3 antibody is a neutralizing human anti-TIM-3 antibody or a humanized neutralizing anti-TIM-3 antibody.