Methods for inhibiting microglial activation

Abstract

The present disclosure provides methods for inhibiting microglial cell activation, ameliorating or treating the neurological effects of cerebral ischemia or inflammation, and ameliorating or treating certain diseases affecting the CNS by intranasal administration of anti-CD3 antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 255,809, filed October 14, 2021, U.S. Provisional Patent Application No. 63 / 315,331, filed March 1, 2022, and U.S. Provisional Patent Application No. 63 / 349,422, filed June 6, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Incorporation by reference of sequence listing The Sequence Listing XML associated with this application has been provided electronically in XML file format and is incorporated herein by reference. The XML filename containing the Sequence Listing XML is "TIZI-034-001WO SeqList ST26". The XML file is 10,747 bytes, was created on October 12, 2022, and has been submitted electronically via the USPTO Patent Center.

[0003] FIELD OF THEINVENTION The present invention relates generally to methods of ameliorating or treating the neurological effects of microglial activation, and to methods of ameliorating or treating certain diseases affecting the CNS, by administering anti-CD3 antibodies. [Background technology]

[0004] The human CD3 antigen consists of at least four invariant polypeptide chains that are noncovalently associated with the T cell receptor on the surface of T cells and are now commonly referred to as the CD3 antigen complex. It is intimately involved in the process of T cell activation in response to antigen recognition by the T cell receptor.

[0005] Due to the fundamental nature of CD3 in the initiation of anti-antigen responses, monoclonal antibodies against this receptor are able to block or at least modulate immune processes and have therefore been proposed as drugs for the treatment of inflammatory and / or autoimmune diseases.

[0006] The central nervous system (CNS) has long been considered a site of relative immune privilege. However, it is increasingly recognized that CNS tissue injury in acute and chronic neurological diseases may be mediated by CNS inflammatory responses. CNS inflammatory responses are primarily mediated by inflammatory cytokines.

[0007] There is a need in the art for more specific therapeutic targeting systems for controlling microglial activation and neuroinflammation. Summary of the Invention

[0008] In one aspect, provided herein is a method of treating or alleviating a sign or symptom of a disease associated with microglial activation in a subject, comprising intranasally administering to the subject a daily dose of about 10 μg to 200 μg of an anti-CD3 antibody. In some embodiments, the disease associated with microglial activation is a neurodegenerative disorder, an ischemia-related disease or injury, a traumatic brain injury, or a lysosomal storage disease. In some embodiments, the neurodegenerative disease is multiple sclerosis (MS), Alzheimer's disease (AD), Lewy body disease, Parkinson's disease (PD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), epilepsy, HIV-associated encephalopathy, or AIDS-associated dementia. In some embodiments, the ischemia-associated disease is ischemia-reperfusion injury, stroke, or myocardial infarction. In some embodiments, the ischemia-reperfusion injury is in lung tissue, cardiac tissue, and neurological tissue. In some embodiments, the traumatic brain injury is a concussion or whiplash injury. In some embodiments, the concussion is repetitive concussion injury. In some embodiments, the lysosomal storage disease is Niemann-Pick disease. In some embodiments, the sign or symptom of a disease associated with microglial activation is amyloid plaque formation.

[0009] In some embodiments, the anti-CD3 antibody is a monoclonal or polyclonal antibody. In some embodiments, the anti-CD3 antibody is fully human, humanized, or chimeric. In some embodiments, the anti-CD3 antibody comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO:1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:3), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO:4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO:5), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO:6), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO:7). In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8, and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0010] In some embodiments, the daily dose is administered once a day. In some embodiments, the daily dose is 50 μg. In some embodiments, the daily dose is divided evenly between each nostril. In some embodiments, the daily dose is administered three times a week. In some embodiments, the daily dose is administered to the subject in at least one cycle, the cycle being once a day, three times a week for two weeks. In some embodiments, the cycle is repeated 2-10 times. In some embodiments, the cycle is followed by a drug holiday. In some embodiments, the drug holiday is for one week.

[0011] In some embodiments, the method results in an improvement in EDSS score in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the EDSS score before administration of the anti-CD3 antibody. In some embodiments, the method results in an improvement in pyramidal score in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the pyramidal score before administration of the anti-CD3 antibody. In some embodiments, the method results in an improvement in walking ability as measured by a 25-foot timed walk test in the subject of at least 2 seconds, at least 3 seconds, at least 5 seconds, at least 10 seconds, at least 15 seconds, or at least 20 seconds, compared to the walking ability before administration of the anti-CD3 antibody. In some embodiments, the method results in a reduction in microglial activation as measured by a PET scan in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the level of microglial activation before administration of the anti-CD3 antibody. In some embodiments, the method results in a decrease in the levels of IL-6, IL-1β, IFN-γ, and / or IL-18 in the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to levels before administration of the anti-CD3 antibody. In some embodiments, the method results in an increase in the level of CD8 naive cells and / or a decrease in CD8 effector cells in the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to levels before administration of the anti-CD3 antibody.

[0012] In another aspect, provided herein is a method of treating or alleviating a sign or symptom of a disease associated with neuroinflammation in a subject, comprising intranasally administering to the subject a daily dose of about 10 μg to 200 μg of an anti-CD3 antibody. In some embodiments, the disease is multiple sclerosis (MS), Alzheimer's disease (AD), Lewy body disease, Parkinson's disease (PD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS).

[0013] In some embodiments, the anti-CD3 antibody is a monoclonal or polyclonal antibody. In some embodiments, the anti-CD3 antibody is fully human, humanized, or chimeric. In some embodiments, the anti-CD3 antibody comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO:1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:3), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO:4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO:5), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO:6), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO:7). In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8, and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0014] In some embodiments, the daily dose is administered once per day. In some embodiments, the daily dose is 50 μg. In some embodiments, the daily dose is divided evenly between each nostril. In some embodiments, the daily dose is administered three times per week. In some embodiments, the daily dose is administered to the subject in at least one cycle, the cycle being once per day, three times per week for two weeks. In some embodiments, the cycle is repeated 2-10 times. In some embodiments, the cycle is followed by a drug holiday. In some embodiments, the drug holiday is for one week. In some embodiments, the method results in a reduction of neuroinflammation in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the level of neuroinflammation prior to administration of the anti-CD3 antibody.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In carrying out the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0016] Other features and advantages of the invention will be apparent from and are included in the following detailed description and claims. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows PET images before and after forualumab treatment. [Diagram 2] Figure 2 shows PET images demonstrating a marked decrease in [F-18]PBR06 uptake in a high affinity binder SPMS subject after 3 months of intranasal forualumab treatment (top row), whereas a test-retest [F-18]PBR06-PET scan performed in another high affinity binder secondary progressive multiple sclerosis (SPMS) subject (who did not receive any therapeutic intervention between scans) showed no significant difference in visual interpretation (bottom row). [Diagram 3] FIG. 3 shows PET images demonstrating a sustained decrease in [F-18]PBR06 uptake in a high affinity binder SPMS subject after 3 months of intranasal forualumab treatment followed by a 7-week washout period. [Figure 4] FIG. 4 shows bar graphs depicting standardized [F-18]PBR06 uptake values ​​(SUV) in the whole brain, cortex, thalamus, white matter and cerebellum at baseline, 3 months and 4.7 months. [Diagram 5]FIG. 5 shows bar graphs depicting standardized [F-18]PBR06 uptake value ratios (SUVR) in the whole brain, cortex, thalamus, white matter and cerebellum at baseline, 3 months and 4.7 months. [Figure 6] FIG. 6 is a chart showing SUV and SUVR in the whole brain, cortex, thalamus, white matter, and cerebellum at baseline, 3 months, and 4.7 months compared to sham reference regions. [Figure 7A] Forualumab stimulation of PBMC in vitro. Figure 7A shows a representation of FACS data showing the frequency of T cells that are CD4+ or CD4 undergoing microcytodilution following 5 days of peripheral blood mononuclear cell (PBMC) stimulation with UCHT1 or forualumab (1 μg / ml) with or without anti-CD28 (0.5 μg / ml) and IL-2 (5 U / ml). [Figure 7B-7C] Figure 7B is a bar graph showing the percentage of expanded T cells from PBMC cultures stimulated with UCHT1 or forualumab that express CD4 (data from three healthy donors). Figure 7C shows a bar graph comparison of the percentage of expanded T cells (i.e., given microdilutions of cells) in CD4 vs. CD8 T cells in the indicated 5-day PBMC cultures. [Figure 7D-7E] Figures 7D-7F show representative FACS plots showing proliferation (cell trace dilution) from forualumab- or UCHT1-stimulated cultures of CD4 T cells alone (Figure 7D) or CD4 and CD8 T cells (Pan T cells, Figure 7E) derived from the same PBMCs, all stimulated in the presence of irradiated, T cell-depleted PBMCs, such as APCs and IL-2 (5 U / ml). [Figure 7F] FIG. 7F shows evidence of potential co-expression of IFNγ and IL-17 in CD4 T cells from different healthy donors stimulated with the indicated anti-CD3 mAbs and IL-2 or IL-2 / anti-CD28. [Figure 7G]Purified CD4 T cells or CD4 and CD8 T cells (derived by pan-T cell isolation of PBMCs) were stimulated with forualumab or UCHT1 (1 μg / ml) in the presence or absence of anti-CD28 (0.5 μg / ml CD28.2 mAb, BD) and IL-2 (5 U / ml) as indicated, and irradiated T cell-depleted PBMCs as APCs (Figure 7G). Significance by One Way ANOVA (with Sidak's correction for multiple comparisons, *p<0.05, **p<0.001). [Figure 8A] Surface CD3 and Volkswagen-treated longitudinal PBMC were stained for CD3 and changes in frequency of CD3 dividing cells or intensity (MFI) of CD3 compared to baseline (T1) levels were measured (Figure 8A). [Figure 8B] Figure 8B shows lineage and differentiation markers in the 10 μg, 50 μg, 250 μg and placebo groups. CD8+ refers to CD8+CD45RA-CD27-CD8 Tem, CD4+CD45RA+CD27+ naive CD4 T, and CD3+CD4-CD8-DN. C, D. Representative dot plots of CD8 TEMRA, naive CD8 T cells, and CD8 T cell GzmB expression in the entire cohort. [Figure 8C] FIG. 8C shows the changes in frequency of naive CD8 T cells (CD45RA+CD27+) and CD8 T cells (CD45RA+CD27-). [Figure 8D] Figure 8D shows the change in ex vivo CD8 T cell expression frequency of GzmB. Within each group, change over time was estimated using a linear mixed-effects model with a fixed categorical effect of time and a random intercept. *p<0.05, **p<0.001). [Figure 9A-9B]RNA-seq analysis of PBMCs from healthy volunteers treated with 50 μg of forualumab. Figure 9A is a graph of single cell analysis of CD8+ populations isolated from PBMCs, showing changes in CD8 maturation stage subsets derived from scRNA data, with cell types defined by clusters based on unbiased DEGs. Figure 9B shows the data in aggregated bar graph or line graph analysis. Different maturation subsets of CD8 T cells show unique DEGs between baseline (T1) and T2. [Figure 9C] Figures 9C-9E are heatmap presentations of genes that showed increased or decreased expression from baseline after forualumab treatment. Gene expression values ​​were used to separate cells into naive CD8 T cells, naive-like CD8 T cells (where expression of the top set of genes differed from naive), intermediate CD8 T cells (which showed characteristics of both naive and memory cells), effector memory, and TEMRA (Figure 9C). [Figure 9D] Using the various mature subset gene expression values ​​of CD4 T cells, the cells were separated into naive CD4 T cells, intermediate CD4 T cells (which exhibit characteristics of both naive and memory cells), memory CD4, and memory CD4 with strong GALS1 gene expression (Figure 9D), which are distinct functional subsets of monocytes. [Figure 9E] Gene expression values ​​were used to separate cells into classical, non-classical, and intermediate subsets (Figure 9E). [Fig. 9F-9G] Figure 9F is a series of violin plots showing the change in expression of TIGIT, TGFb1 and KIR3DL2 in CD8 effector memory and CD8 TEMRA cells. Figure 9G is a series of violin plots showing the change in expression of CTLA4, KLRG1 and TGFb1 in naive and memory CD4 T cells. Significance scores posted for change from baseline were determined using unpaired two-tailed T-tests for time point 1 (T1vT2, T1vT3, T1vT4). *p<0.05, **p<0.001, ***p<0.0001). [Figure 10A-10C]Figures 10A and B. Serum IgG and IgM antibody reactivity in patients treated with 50 μg forualumab. Heat maps depicting mean IgG (Figure 10A) and IgM (Figure 10B) antibody reactivity in serum samples from patients. Figure 10C is a volcano plot depicting differential IgG and IgM antibody reactivity. Cutoff criteria were defined as p-value <0.05 and log2 fold change >1 or <-1. [Figure 11] FIG. 11 shows patient predisposition. [Figure 12] Figure 12 shows Treg heatmap. DEGs observed between baseline (T1) and T2 in CD4+CD25hi CD127low Tregs are shown for naive and activated (memory) Tregs. [Figure 13] Figures 13A-13D show the relationship between differentially expressed genes in CD8+ TEMRA and immune function. The functional characteristics of the identified DEGs were obtained from the literature and assigned to one of 12 groups (Figures 13A and 13B). Figure 13C shows genes that were downregulated from T1 to T2 after treatment. Figure 13D shows genes that were upregulated from T1 to T2 after treatment. Dark gray indicates a pro-inflammatory role on a specific immune function. Light gray indicates an anti-inflammatory role on a specific immune function. As shown in Figure 13C, 17 / 19 genes with pro-inflammatory roles were downregulated by treatment, while in Figure 13D, 16 / 24 genes with anti-inflammatory roles were upregulated. [Figure 14] 14 shows PET images showing normalized uptake ratio images in patient 2 (EA2) demonstrating a significant decrease in [F-18]PBR06 uptake after 3 months of treatment with intranasal forualumab, with widespread reductions in the cortex, thalamus, white matter and cerebellum. [Figure 15] FIG. 15 is a graph showing the decrease in PET signal in EA2 after 3 months of treatment with intranasal foruarumab. [Figure 16] FIG. 16 shows the EDSS scores for patient 2 (EA2). [Figure 17]FIG. 17 is a graph showing the 25 foot timed walk for patient 2 (EA2). [Figure 18] FIG. 18 is a graph showing EDSS and pyramidal scores over time in patient 1 (EA1). [Figure 19] FIG. 19 is a graph showing the time it took Patient 1 to walk 25 feet over time. [Figure 20A] Figures 20A and 20B show the level of microglial activation over time in patient 1. Figure 20A shows PET images. [Figure 20B] FIG. 20B shows quantification of activated microglial cell PET signal (SUVR-1). [Figure 21A] 21A-21D show the levels of IL-6, IL-1β, INF-γ, and IL-18, respectively, over time in patient 1. [Figure 21B] 21A-21D show the levels of IL-6, IL-1β, INF-γ, and IL-18, respectively, over time in patient 1. [Figure 21C] 21A-21D show the levels of IL-6, IL-1β, INF-γ, and IL-18, respectively, over time in patient 1. [Figure 21D] 21A-21D show the levels of IL-6, IL-1β, INF-γ, and IL-18, respectively, over time in patient 1. [Figure 22] FIG. 22 is a graph showing EDSS and pyramidal scores over time in patient 2 (EA2). [Diagram 23] FIG. 23 is a graph showing the time it took Patient 2 to walk 25 feet over time. [Figure 24A] Figures 24A-I. Intranasal anti-CD3 improves pathological outcomes in a CCI model of TBI. Figure 24A: Experimental timeline of intranasal anti-CD3 treatment and visual depicting time points after CCI (asterisks) and histopathological experiments performed. [Figure 24B] FIG. 24B: Brain edema was analyzed 3 days after TBI, and % water content was measured between ipsilateral and contralateral hemispheres by Student's t-test. [Figure 24C] (C) Magnetic resonance imaging (MRI) was then performed using a 3-Tesla MRI to measure parenchymal lesion volume at day 7 after CCI. Sequential MRI images were taken of TBI-aCD3 and TBI-Iso at day 7 after CCI. [Figure 24D] FIG. 24D: MRI lesion volumes 7 days after TBI were analyzed by Student's t-test. [Figure 24E] FIG. 24E: Brain sections were stained with hematoxylin and eosin (H&E) 1 month after TBI, and lesion volumes were measured by Image J software and analyzed by Student's t-test. [Figure 24F] Figure 24F: CD11b+ Ly6Chi classical monocytes were examined from the ipsilateral hemisphere of the brain by fluorescence-activated cell sorting (FACS) among Sham-Iso, TBI-Iso, and TBI-aCD3 groups on day 5 after TBI. Statistical analysis by one-way ANOVA followed by Tukey post-hoc analysis. [Figure 24G] FIG. 24G: Brain sections 1 month after TBI were stained with Iba-1 antibody and co-stained with DAPI, and the % area covered by Iba-1 positive cells was quantified by Image J and analyzed by one-way ANOVA followed by Tukey post hoc analysis. [Fig. 24H] Figure 24H: Brain sections 7 days after TBI were stained with TUNEL and co-stained with 7-AAD and DAPI. Representative images were taken at 20x magnification in the peri-contusion cortex. Five sections of each sample were prepared to capture the peri-contusion area, and the number of TUNEL-positive cells around the peri-contusion was quantified by Image J and analyzed by Student's t-test. [Figure 24I]Figure 24I: Total CD4+, CD4+ Foxp3+, and CD4+ LAP+ Tregs were examined from the ipsilateral hemisphere of the brain and cervical lymph nodes by fluorescence-activated cell sorting (FACS) at 7 days after TBI among Sham-Iso, TBI-Iso, and TBI-aCD3 groups. Statistical analysis by one-way ANOVA followed by Tukey post-hoc analysis. n=4-5 mice / group were used for all experiments and data are shown as (mean and SEM). *P<0.05, **P<0.01, ***P<0.001, ns=not significant. Scale bars are 1 mm (Figures 24G and 24E) and 500 μm (Figure 24H) for whole tissue sections. Peri-contusion area of ​​200 μm is 100 μm when zooming in on sections. (g) All images were taken at 20x magnification. [Figure 25A] Nasal anti-CD3 improves behavioral outcomes in a CCI model of TBI. Figure 25A: Visual depicting treatment regimens in a moderate CCI model of TBI (depth: 1 mm, impact tip diameter: 1.5 mm) with both early and delayed intranasal anti-CD3 regimens. [Figure 25B] Figures 25B and 25C: Behavioral tests measured in the open field, rotarod, Morris water maze, probe test, anxiety-like behavior, and locomotor activity were assessed among Sham-Iso, TBI-Iso, and TBI-aCD3 groups in two independent cohorts undergoing early and delayed treatment regimens. [Figure 25C] Figures 25B and 25C: Behavioral tests measured in the open field, rotarod, Morris water maze, probe test, anxiety-like behavior, and locomotor activity were assessed among Sham-Iso, TBI-Iso, and TBI-aCD3 groups in two independent cohorts undergoing early and delayed treatment regimens. [Figure 25D] Figure 25D: Visual depicting the timeline of the treatment regimen for a severe traumatic brain injury CCI model (depth: 1.5 mm, impact tip diameter: 3.0 mm) with early intranasal anti-CD3 therapy. [Figure 25E]Figure 25E: Behavioral tests of locomotor activity measured by rotarod, Morris water maze, probe test, anxiety-like behavior, and open field were evaluated among Sham-Iso, TBI-Iso, and TBI-aCD3 groups. Morris water maze was analyzed by two-way ANOVA, while the other behavioral tests were analyzed by one-way ANOVA followed by Tukey post-hoc analysis. n=8-12 mice / group were used for all experiments, and data are presented as (mean and SEM). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant. [Figure 25F] FIG. 25F: Relative expression of microglia, astrocyte, oligodendrocyte, and neuronal markers by bulk RNA-seq in Sham-Iso animals (n=5) from which 4D4+ microglia were isolated. [Figure 25G] Figure 25G: Significantly upregulated GO Biological Process (BP) pathways for TBI-aCD3 vs. Sham-Iso and TBI-Iso vs. Sham-Iso at day 7. Data are expressed as log2 fold change. Only significant GOBP pathways (P<0.05) from comparison (TBI-aCD3 vs. Sham-Iso or TBI-Iso vs. Sham) are shown. Pathway analysis was performed using GAGE. [Fig. 25H] FIG. 25H: Visual depiction of the experimental timeline for in vivo phagocytic function studies (FIG. 26I). [Figure 26A] Figures 26A-26L: Intranasal anti-CD3 modulates acute and chronic microglial responses following TBI. Figure 26A: Images representing two independent experiments showing 4D4+ ly6C microglia isolation and bulk RNA sequencing 7 days and 1 month after TBI and intranasal anti-CD3 treatment. [Figure 26B] (B) DiVenn plot showing uniquely shared differentially expressed genes (P<0.05) between TBI-Iso vs. TBI-Iso. Sham-Iso and TBI-aCD3 vs. Sham-Iso groups at both 1 week and 1 month after TBI. The directionality of gene expression is determined by the log2-fold change in pairwise gene expression. [Figure 26C-1]FIG. 26C: Top 1000 differentially expressed genes (P<0.05) across all different groups (Sham-Iso, TBI-Iso, and TBI-aCD3) for each time point: 7 days (left panel) and 1 month (right panel). [Figure 26C-2] FIG. 26C: Top 1000 differentially expressed genes (P<0.05) across all different groups (Sham-Iso, TBI-Iso, and TBI-aCD3) for each time point: 7 days (left panel) and 1 month (right panel). [Figure 26D] Figure 26D: Significantly upregulated GO Biological Process (BP) pathways for TBI-aCD3 vs. Sham-Iso and TBI-Iso vs. Sham-Iso at 1 month. Data are expressed as log2 fold change. Only significant GOBP pathways (P<0.05) from comparison (TBI-aCD3 vs. Sham-Iso or TBI-Iso vs. Sham-Iso) are shown. Pathway analysis was performed using GAGE. [Figure 26E-1] Figure 26E: Significant genes encoding the microglial core sensome in the TBI-aCD3 group compared to the TBI-Iso group at either day 7 or month 1 (right panel). Differentially expressed genes at each time point are in bold and indicated with an asterisk (P<0.05). Each gene is color-coded based on its function in the microglial core sensome. A preselected list of microglial sensome genes was previously published (Faul et al. Handb Clin Neurol 127, 3-13 (2015)). [Figure 26E-2] FIG. 26E: Genes encoding microglial core sensomes in the TBI-aCD3 group compared to the TBI-Iso group at day 7 (left panel) and 1 month (right panel) were significant at either day 7 or month 1. [Figure 26F-1] FIG. 26F: Heatmap showing relative expression levels of genes at two time points (7 days and 1 month) involved in the following pathways: phagocytosis and proinflammatory response. [Figure 26F-2]FIG. 26F: Heatmap showing relative expression levels of genes at two time points (7 days and 1 month) involved in the following pathways: phagocytosis and proinflammatory response. [Figure 26G] Figures 26F and 26G: Heatmaps showing the relative expression levels of genes at two time points (7 days and 1 month) involved in the following pathways: phagocytosis and pro-inflammatory response. [Fig. 26H] Figure 26H: DAM / MGnD heatmap at 1 month showing relative expression levels of genes. Bold genes with asterisks (*) indicate differentially expressed genes (P<0.05). A preselected set of genes part of the DAM / MGnd pathway, proinflammatory pathway, and phagocytic pathway were extracted from the literature (Krasemann et al. Immunity 47, 566-581 e569 (2017); Keren-Shaul et al. Cell 169, 1276-1290 e1217 (2017)). [Figure 26I] FIG. 26I: In vivo phagocytic function experiments in which mice were injected with either labeled apoptotic neurons or DPBS. [Figure 26J] Gating strategy showing phagocyte positive microglia in TBI-Iso and TBI-aCD3 animals. A visual representation of the experimental timeline of the study can be seen in Figure 25H. Data are shown as violin plots, with n=5 animals per group. Animals receiving DPBS are not shown in Figure 26I. Bar plots of quantitative PCR of the ipsilateral hemisphere at 7 days and 1 month after TBI. Expression was normalized to GAPDH and shown relative to that of Sham-Iso animals. Significant expression of IL-10 (7 days), IL6, IFNg, Tnf, IL17, Bdnf (1 month) was shown. N=6-8 mice / group (mean and SEM). Statistical analysis by one-way ANOVA followed by Tukey post hoc analysis. *P<0.05, ns=not significant. [Figure 26K-1] Quantitative PCR bar graphs of significant (FIG. 26K) and non-significant cytokines (FIG. 26L) in the ipsilateral hemisphere among Sham-Iso, TBI-Iso, and TBI-aCD3 groups at 7 days and 1 month after TBI. [Figure 26K-2] Quantitative PCR bar graphs of significant (FIG. 26K) and non-significant cytokines (FIG. 26L) in the ipsilateral hemisphere among Sham-Iso, TBI-Iso, and TBI-aCD3 groups at 7 days and 1 month after TBI. [Figure 26L-1] Quantitative PCR bar graphs of significant (FIG. 26K) and non-significant cytokines (FIG. 26L) in the ipsilateral hemisphere among Sham-Iso, TBI-Iso, and TBI-aCD3 groups at 7 days and 1 month after TBI. [Figure 26L-2] Quantitative PCR bar graphs of significant (FIG. 26K) and non-significant cytokines (FIG. 26L) in the ipsilateral hemisphere among Sham-Iso, TBI-Iso, and TBI-aCD3 groups at 7 days and 1 month after TBI. [Figure 26L-3] Quantitative PCR bar graphs of significant (FIG. 26K) and non-significant cytokines (FIG. 26L) in the ipsilateral hemisphere among Sham-Iso, TBI-Iso, and TBI-aCD3 groups at 7 days and 1 month after TBI. [Figure 26L-4] Quantitative PCR bar graphs of significant (FIG. 26K) and non-significant (FIG. 26L) cytokines in the ipsilateral hemisphere among Sham-Iso, TBI-Iso, and TBI-aCD3 groups at 7 days and 1 month after TBI. Expression was normalized to GAPDH. n=6-8 mice / group (mean and SEM). Statistical analysis by one-way ANOVA followed by Tukey post-hoc analysis. *P<0.05, **P<0.01, ***P<0.001, ns=not significant. [Figure 27] Intranasal anti-CD3 improves behavioral outcomes after TBI in an IL-10-dependent manner. [Figure 27A] Figure 27A: Heatmap shows the relative expression of IL-10 pathway genes at 7 days and 1 month after CCI in Sham-Iso, TBI-Iso, and TBI-aCD3 groups. The IL-10 pathway gene list was taken from Xue et al. Immunity 40, 274-288 (2014). [Figure 27B]Figure 27B: IL-10 expression was analyzed by flow cytometry in CD4+, FoxP3+, FoxP3-, LAP+, LAP-, FCRLS+ microglia (Butovsky et al. Nat Neurosci 17, 131-143 (2014)), NK1.1+, Ly6C+, and Ly6Gh subpopulations 7 days after TBI and intranasal anti-CD3 treatment. N=4-5 mice / group (mean+SEM). Statistical analysis by one-way ANOVA followed by Tukey post hoc analysis. [Figure 27C] Figure 27C: Visual representation of the experimental timeline of intranasal anti-CD3 and anti-IL-10 receptor blocking mAb (aIL-10R) treatment and time points after CCI (stars) and behavioral studies performed. Anti-IL-10 receptor blocking mAb (aIL-10R) (0.5 mg / mouse) is injected intraperitoneally every 3 days (black arrows). [Figure 27D-1] FIG. 27D: Behavioral tests of locomotor activity measured by rotarod, Morris water maze, probe test, anxiety-like behavior, and open field were assessed in Sham-Iso, TBI-Iso, TBI-aCD3, and TBI-aCD3-anti-IL10 groups. [Figure 27D-2] Morris water maze was analyzed by two-way ANOVA and other behavioral tests by one-way ANOVA followed by Tukey post-hoc analysis, n=8 mice / group. [Figure 27E] FIG. 27E: Top 1000 differentially expressed genes (P<0.05) across all different groups (Sham, TBI-Iso, TBI-aCD3, and TBI-aCD3-aIL10R). [Figure 27F]Figure 27F: GO Biological Process (BP) pathways significantly down- or upregulated for TBI-aCD3 vs. Sham-Iso, TBI-Iso vs. Sham-Iso, and TBI-aCD3-aIL10R vs. Sham-Iso. Sham-Iso (gold) at 1 month. Data are expressed as log2 fold change. Only GOBP pathways significant (P<0.05) from all three comparisons (TBI-aCD3 vs. Sham-Iso, TBI-Iso vs. Sham, or TBI-aCD3-aIL10R vs. Sham-Iso) are shown. Pathway analysis was performed using GAGE. [Figure 27G] FIG. 27G: Visual representation of microglia and Treg transwell co-culture. 4D4+ ly6C- microglia were isolated 24 hours after TBI and cultured for 7 days. At the same time, different cohorts of TBI animals were treated with intranasal aCD3 or isotype control for 7 days, and their splenic total CD4+ cells were sorted and placed on top of the microglia transwell culture. After leaving the transwell system for 72 hours, microglia were lysed and analyzed by quantitative PCR. [Fig. 27H] Figure 27H: Quantitative PCR heatmap of microglia from TBI-Iso total CD4 and TBI-aCD3 total CD4, where expression was normalized to GAPDH and analyzed by Student's t-test. n=3 wells per group, with 200,000 microglial cells in each well. Total CD4+ in each insert was 800,000 cells pooled from 3 biological replicate animals. Expression of Il10, Tgfb1, CD206, and CD14 is shown as bar graphs (mean and SEM). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=not significant. [Figure 27I] FIG. 27I: DiVenn plot showing unique and shared microglial differentially expressed genes (P<0.05) between TBI-Iso pairs. Comparison of Sham-Iso, TBI-aCD3 vs. Sham-Iso, and TBI-aCD3+aIL10R vs. Sham-Iso groups. Directionality of gene expression is determined by the log2 fold change of pairwise gene expression. [Figure 27J-1] FIG. 27J: Quantitative PCR bar plot of microglia between TBI-Iso total CD4+ and TBI-aCD3 total CD4+ after 72 hours of transwell co-culture (FIG. 27G); expression was normalized to GAPDH and analyzed by Student's t-test (mean and SEM). [Figure 27J-2] n=3 wells per group with 200,000 microglial cells in each well. In each insert, a total of 800,000 CD4+ cells were pooled from 3 biological replicate animals. ns=not significant. [Figure 28A-1] CD4+FoxP3+ regulatory T cells improve microglial responses and behavioral outcomes after TBI. Figure 28A: Visual depiction of the experimental timeline of the adoptive transfer experiment. [Figure 28A-2] Splenic CD4+ from isotype and aCD3-treated animals (CD45.2) and CD4+FoxP3GFP- populations from aCD3-treated animals (CD45.2) 7 days after TBI were transferred intraperitoneally into untreated but CCI-injured (CD45.1) mice. Adoptive transfers were performed at three different time points, with each animal receiving 2.5 million cells per injection. [Figure 28B] Figure 28B: Behavioral tests of rotarod, Morris water maze, probe test, anxiety-like behavior, and locomotor activity (measured by open field) were evaluated among WT Sham (DPBS-treated, baseline), Iso-total CD4+, aCD3-total CD4+, and aCD3-FoxP3(-)GFP groups. Morris water maze was analyzed by two-way ANOVA, the other behavioral tests by one-way ANOVA followed by Tukey post-hoc analysis. n=8 mice / group. [Figure 28C] Figure 28C: Visual representation of an independent experiment in which splenic CD4+ cells from 7-day treated TBI (CD45.2) animals were injected intraperitoneally into untreated but CCI injured (CD45.1) animals and %CD45.2 cells were analyzed by fluorescence activated cell sorting (FACS) 3 days after injection. [Figure 28D]Figure 28D: Flow cytometry gates of brain, cervical lymph nodes, and spleens of (CD45.1) animals showing the percent of CD45.2 cell infiltration, n=5 mice / group, brains were pooled from 5 ipsilateral hemispheres. [Figure 28E] Figure 28E: DiVenn plot showing specific and shared differentially expressed microglial genes (P<0.05) between aCD3-total CD4+ pairs. Total CD4+ and aCD3-FoxP3(-)GFP vs. Iso-total CD4+. The directionality of gene expression is determined by the log2-fold change of pairwise gene expression. [Figure 28F] Figure 28F: Top 1000 differentially expressed genes across Iso-total CD4+, aCD3-FoxP3(-)GFP, and aCD3-total CD4+ groups (P<0.05). [Figure 28G] Figure 28G: GO biological process pathways are significantly down- or up-regulated in aCD3-total CD4+ versus Iso-total CD4+ (red) and aCD3-FoxP3(-)GFP versus Iso-total CD4+. Data are expressed as log2 fold change. Only significant GOBP pathways from comparison (aCD3-total CD4+ versus Iso-total CD4+ or aCD3-FoxP3(-)GFP versus Iso-total CD4+) are shown (P<0.05) Iso-total CD4+). Pathway analysis was performed using GAGE. [Fig. 28H] FIG. 28H: Bar plot of quantitative PCR of ipsilateral hemisphere between Iso-total CD4+, aCD3-FoxP3(-)GFP group, and aCD3-total CD4+; expression normalized to GAPDH. Expression of IL-10, Tgfb1, Il2, Il22, and Gdnf 1 month after TBI and three adoptive transfer experiments are shown. n=5 mice / group (mean+SEM). Statistical analysis by one-way ANOVA followed by Tukey post hoc analysis. *P<0.05, **P<0.01, ns=not significant. [Figure 28I-1] Figure 28I: Quantitative PCR bar plots show cytokine expression in the ipsilateral hemisphere of aCD3-Total CD4+, aCD3-FoxP3(-)GFP, and Iso-Total CD4+ adoptive transfer experimental groups at 1 month post-TBI. [Figure 28I-2]Figure 28I: Quantitative PCR bar plots show cytokine expression in the ipsilateral hemisphere of aCD3-total CD4+, aCD3-FoxP3(-)GFP, and Iso-total CD4+ adoptive transfer experimental groups at 1 month after TBI. n=5 mice / group (mean and SEM). *P<0.05, ns=not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The methods and compositions described herein are based in part on the discovery that the inflammatory phenotype of microglial cells is modulated by anti-CD3 antibodies. Specifically, it was discovered that CD74, an invariant chain involved in MHC II presentation, and H2-AB1, an MHC II antigen, are downregulated in microglia upon anti-CD3 administration. Importantly, anti-CD3 administration not only modulated gene expression of Clec7+ microglia in APPPS1 mice, but also reduced the number of Clec7+ plaque-associated microglia.

[0019] More specifically, the methods described herein relate to reducing microglial activation by decreasing CD3 expression.

[0020] Microglia are non-neuronal macrophage-like cells present in the developing and adult central nervous system. Upon neuronal injury, microglia are converted from a resting to an activated state, characterized by changes in morphology, immunophenotype, migration, and proliferation. Activated microglia are involved in the phagocytosis of neurons, and microglial proteases are involved in the degradation of neurons.

[0021] The methods and compounds are useful for preventing, treating, or ameliorating neurological signs and symptoms associated with chronic neurological diseases, including, but not limited to, multiple sclerosis (MS), Alzheimer's disease (AD), Lewy body disease, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), epilepsy, HIV-associated encephalopathy, and AIDS-associated dementia.

[0022] The methods are also useful for preventing, treating, or ameliorating neurological signs and symptoms associated with inflammatory conditions affecting the nervous system, including the CNS.

[0023] In other words, the methods and compounds are useful for preventing, suppressing, or reducing microglial activation in the CNS that occurs as part of an acute or chronic CNS disease. Suppression or reduction of microglial activation can be assessed by a variety of methods, as will be apparent to those skilled in the art; one such method is to measure the production or presence of compounds known to be produced by activated microglia and compare such measurements with the levels of the same compounds in a control situation. Alternatively, the effectiveness of the methods and compounds in suppressing, reducing, or preventing microglial activation can be assessed by comparing the signs and / or symptoms of the CNS disease in treated and control subjects, where such signs and / or symptoms are associated with or secondary to microglial activation.

[0024] As used herein, the terms "combat," "treat," and "ameliorate" are not necessarily meant to indicate a reversal or halt of the disease process underlying the CNS condition afflicting the subject being treated. Such terms indicate that adverse signs and / or symptoms associated with the condition being treated are reduced or diminished, or progress at a slower rate, compared to those that would occur if not treated. Changes in disease signs or symptoms may be assessed at the subject level (e.g., the subject's function or condition is assessed), or at the tissue or cellular level (e.g., production of a marker of glial activation is reduced or diminished). When the methods disclosed herein are used to treat chronic CNS conditions (such as multiple sclerosis, or MS), the methods do not necessarily affect or reverse the underlying disease process, but may delay or delay the onset of symptoms.

[0025] Surprisingly, intranasal forualumab was safe at doses of 10 μg, 50 μg, and 250 μg administered for 5 consecutive days to healthy subjects. Immune effects were primarily observed at the 50 μg dose. The dose effect of 50 μg being more immunomodulatory than 250 μg is consistent with animal studies of mucosal immune tolerance where higher doses do not induce immunomodulation, most likely due to partial signaling occurring at intermediate doses that favor the induction of regulatory cells. Importantly, the biological effects of intranasal anti-CD3 are significantly different from those occurring with intravenous anti-CD3. IV anti-CD3 has been associated with modulation of CD3 from the cell surface, side effects including CD3 cell depletion and cytokine release syndrome, and in some cases, activation of EBV. EBV reactivation was observed with IV forualumab at doses of 500 μg and 1000 μg. In contrast, no EBV activation was observed with intranasal forualumab at either dose or modulation of CD3 from the cell surface. Furthermore, when administered intranasally, forualumab was not detectable in the bloodstream. Thus, unlike intravenously administered anti-CD3, which acts systemically by lysing T cells from CD4+ and subsequently reconstituting immunity, nasally administered anti-CD3 acts locally on mucosal surfaces as an immunomodulator. In animal studies, nasally administered anti-CD3 was localized to cervical lymph nodes, and similar to human studies, nasally administered anti-CD3 was not detectable in the bloodstream of animals.

[0026] The in vitro activation properties of forualumab were compared with those of the commonly used anti-CD3 monoclonal antibody UCHT1. + Selectively expand T cells and CD4 + Suppressed T cell proliferation. Purified CD4 + Stimulation of T cells with forualumab increased CTLA4 expression.

[0027] Animal studies have shown that intranasal anti-CD3 induces LAP+ IL-10 secreting Tregs capable of adoptively transferring protection. However, human studies have not shown a significant increase in IL-10. The 50 μg dose resulted in an increase in DN LAP+ T cells at T4. The main effect of intranasal forualumab was the upregulation of CD8+ This occurs in T cells, which is consistent with effects observed with other anti-CD3 monoclonal antibodies administered intravenously in humans. + Decrease in effector memory cells, naive CD8 + and CD4 + Cell expansion and CD8 + A decrease in T cell granzyme B and perforin expression was observed. Antigen array studies also showed the most pronounced effect at the 50 μg dose.

[0028] scRNaseq analysis of subjects receiving the 50 μg dose allowed for a more detailed analysis of the immune effects of intranasal forualumab. Although some of the DEGs functioned in homeostatic cell biology processes, most of the affected DEGs had immunological functions. CD8 + In the CD8+ / CD8+ population, the inflammatory response was anti-inflammatory. Interestingly, we observed an upregulation of TIGIT associated with intravenous administration of teplizumab. + In the TEMRA population, KIR3DL2 was induced in addition to TIGIT, KLTG1, and TGFB1. A similar trend was observed in the non-regulatory CD4 + It was also observed in T cells. + CTLA4 and TGFB1 were upregulated in memory cells, consistent with the results observed after T cell stimulation with forualumab.Only minor changes were observed in the Treg population, with only four DEGs identified, including decreased expression of JUNB, which may enhance Treg stability by inhibiting Th17 differentiation.

[0029] Thus, it does not appear that intranasal forualumab directly expands classical Tregs. Changes were also observed in monocyte populations, including expression of DQ and DP, which are associated with T cells producing higher levels of IL-10. In summary, intranasal anti-CD3 has a strong immunomodulatory effect on the immune response that is dose-dependent, reduces inflammation, and promotes regulation. In summary, intranasal forualumab is safe and induces immune effects when administered at a dose of 50 μg for 5 consecutive days.

[0030] Anti-CD3 antibody The anti-CD3 antibody can be any antibody specific for CD3. The term "antibody" as used herein refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., an antigen-binding portion. For example, examples of immunologically active portions of immunoglobulin molecules include scFv, F(ab) and F(ab')2 fragments that retain the ability to bind to CD3. Such fragments can be obtained commercially or using methods known in the art. For example, F(ab)2 fragments can be generated by treatment with pepsin, a non-specific endopeptidase that usually produces one F(ab)2 fragment and multiple small peptides of the Fc portion. The resulting F(ab)2 fragment is composed of two disulfide-linked Fab units. The Fc fragment can be extensively degraded and separated from the F(ab)2. F(ab) fragments can be generated using papain, a non-specific thiol endopeptidase that, in the presence of a reducing agent, digests IgG molecules into three fragments of similar size: two Fab fragments and one Fc fragment. If Fc fragments are of interest, papain is the enzyme of choice since it produces an Fc fragment of 50,000 daltons; to isolate F(ab) fragments, the Fc fragment can be removed by affinity purification, for example, using protein A / G. Many kits are commercially available for generating F(ab) fragments, including ImmunoPure IgG1 Fab and F(ab')2 preparation kits (Pierce Biotechnology, Rockford, IL). Additionally, commercial services for generating antigen-binding fragments (e.g., Bio Express, West Lebanon, NH) can be used.

[0031] The antibody can be polyclonal, monoclonal, recombinant, e.g., chimeric, deimmunized or humanized, fully human, non-human, e.g., murine, single chain or single domain antibody. The antibody can be of any class, e.g., IgG, IgM, IgA, IgE or IgD. It can also be of any subclass, e.g., IgG1, IgG2, IgG3 and IgG4 or others. Furthermore, in humans, the light chain can be a kappa or lambda chain. In some embodiments, the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind Fc receptors. For example, the anti-CD3 antibody can be an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, e.g., it has a mutagenized or deleted Fc receptor binding region. The antibody can be conjugated to a toxin or imaging agent.

[0032] Many anti-CD3 antibodies are known, including, but not limited to, OKT3 (muromab / Orthoclone OKT3™, Ortho Biotech, Raritan, NJ; U.S. Patent No. 4,361,549); hOKT3 (1 (Herold et al., NEJM 346(22):1692-1698 (2002); HuM291 (Nuvion™, Protein Design Labs, Fremont, Calif.); gOKT3-5 (Alegre et al., J. Immunol. 148(11):3461-8 (1992); 1F4 (Tanaka et al., J. Immunol. 142:2791-2795 (1989)); G4.18 (Nicolls et al., Transplantation 55:459-468 (1999)); (1993)); 145-2C11 (Davignon et al., J. Immunol. 141(6):1848-54 (1988)); Frenken et al., Transplantation 51(4):881-7 (1991); U.S. Patent Nos. 6,491,9116, 6,406,696, and 6,143,297).

[0033] Methods for producing such antibodies are also known. Full-length CD3 protein or antigenic peptide fragments of CD3 can be used as immunogens, as described in U.S. Patent Nos. 4,361,549 and 4,654,210, or can be used to identify anti-CD3 antibodies produced with other immunogens, such as cells, membrane preparations, etc. (e.g., E-rosette positive purified normal human peripheral T cells). Anti-CD3 antibodies can bind to epitopes of any domain or region on CD3.

[0034] Chimeric, humanized, deimmunized, or fully human antibodies may be desirable for applications involving repeated administrations, such as therapeutic treatment of human subjects.

[0035] Chimeric antibodies contain parts of two different antibodies, typically antibodies of two different species. Generally, such antibodies contain a human constant region and a variable region from another species, e.g., a mouse variable region. For example, mouse / human chimeric antibodies have been reported that exhibit the binding characteristics of the parent mouse antibody and the effector functions associated with the human constant region. See, for example, Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No. 4,816,397, all of which are incorporated herein by reference. Generally, these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from existing mouse hybridomas (Nishimura et al., Cancer Research, 47:999 (1987)). The library is then screened for variable region genes from both heavy and light chains that exhibit the correct antibody fragment rearrangement pattern. Alternatively, a cDNA library is prepared from RNA extracted from the hybridoma and screened, or the variable regions are obtained by polymerase chain reaction. The cloned variable region genes are then ligated into an expression vector containing a cloned cassette of the appropriate heavy or light chain human constant region gene. The chimeric genes can then be expressed in a selected cell line, such as a mouse myeloma line. Such chimeric antibodies have been used for human therapy.

[0036] Humanized antibodies are known in the art. Typically, "humanization" results in less immunogenic antibodies that fully retain the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its binding site must be faithfully reproduced in the "humanized" version. This can potentially be achieved by grafting the binding site of a non-human antibody onto a human framework, (a) by grafting the entire non-human variable domain onto a human constant region to create a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci., USA 81:6801 (1984); Morrison and Oi, Adv. Immunol. 44:65 (1988), preserving the ligand binding properties but also retaining the immunogenicity of the non-human variable domain); (b) by grafting only the non-human CDRs onto a human framework and constant region, with or without retention of critical framework residues (Jones et al. Nature, 321:522 (1986); Verhoeyen et al., Science 239:1539 (1989)). (1988)); or (c) by not only grafting entire non-human variable domains (to preserve ligand-binding properties) but also "covering" them with a human-like surface through judicious substitution of exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (1991)).

[0037] Humanization by CDR grafting typically involves grafting only CDRs onto human fragments on human frameworks and constant regions. In theory, this should virtually eliminate immunogenicity (except in the presence of allotypic or idiotypic differences). However, it has been reported that some framework residues of the original antibody must also be preserved (Riechmann et al., Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10,029 (1989)). Framework residues that need to be preserved can be identified by computer modeling. Alternatively, important framework residues can potentially be identified by comparing known antibody binding site structures (Padlan, Molec. Immun. 31(3):169-217 (1994)). The present disclosure also includes partially humanized antibodies in which the six CDRs and a limited number of structural amino acids of the heavy and light chains of a murine monoclonal antibody have been recombinantly grafted onto a CDR-depleted human IgG scaffold (Jones et al., Nature 321:522-525 (1986)).

[0038] Deimmunized antibodies are made by replacing immunogenic epitopes in mouse variable domains with benign amino acid sequences, resulting in deimmunized variable domains that are genetically linked to human IgG constant domains to obtain deimmunized antibodies (Biovation, Aberdeen, Scotland).

[0039] The anti-CD3 antibody can also be a single chain antibody. Single chain antibodies (scFV) can be engineered (see, e.g., Colcher et al., Ann. NY Acad. Sci. 880:263-80 (1999); Reiter, Clin. Cancer Res. 2:245-52 (1996)). Single chain antibodies can be dimerized or multimerized to create multivalent antibodies with specificity for different epitopes of the same target CD3 protein. In some embodiments, the antibody is monovalent, e.g., as described in Abbs et al., Ther. Immunol. 1(6):325-31 (1994), which is incorporated herein by reference.

[0040] An exemplary anti-CD3 antibody comprises heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO:1), heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO:3), heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO:4), light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO:5), light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO:6), and light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO:7).

[0041] In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 8), and a variable light chain amino acid sequence comprising EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 9).

[0042] Preferably, the anti-CD3 antibody is QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP The heavy chain amino acid sequence comprises: QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10), and the light chain amino acid sequence comprises: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11). This anti-CD3 antibody is referred to herein as NI-0401, forualumab, or 28F11AE (see, e.g., Dean Y, Depis F, Kosco-Vilbois M. "Combination therapies in the context of anti-CD3 antibodies for the treatment of autoimmune diseases", Swiss Med Wkly. (2012), the contents of which are incorporated herein by reference in their entirety).

[0043] In some embodiments, the anti-CD3 antibody is a fully human antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody formulation comprises a full-length anti-CD3 antibody. In alternative embodiments, the anti-CD3 antibody formulation comprises an antibody fragment that specifically binds to CD3. In some embodiments, the anti-CD3 antibody formulation comprises a combination of a full-length anti-CD3 antibody and an antigen-binding fragment that specifically binds to CD3.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof that binds CD3 is a monoclonal antibody, a domain antibody, a single chain, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or antigen-binding fragment thereof that binds CD3 is a murine, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0045] Optionally, the anti-CD3 antibody or antigen-binding fragment thereof used in the formulation of the present disclosure comprises at least one amino acid mutation. Typically, the mutation is in the constant region. The mutation results in an antibody with modified effector function. The effector function of the antibody is altered by changing, i.e., increasing or decreasing, the affinity of the antibody for effector molecules such as Fc receptors or complement components. For example, the mutation results in an antibody that can reduce cytokine release from T cells. For example, the mutation is at amino acid residues 234, 235, 265, or 297 in the heavy chain, or a combination thereof.

[0046] Preferably, the mutation results in an alanine residue at any of positions 234, 235, 265 or 297, or a glutamic acid residue at position 235, or a combination thereof.

[0047] Preferably, the anti-CD3 antibodies provided herein contain one or more mutations that prevent the in vivo release of one or more cytokines mediated by the heavy chain constant region.

[0048] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof used in the formulation of the present disclosure is a fully human antibody. A fully human CD3 authenticator as used herein includes, for example, a L234 L235→A234 E235 mutation in the Fc region, which allows cytokine release to be significantly reduced or eliminated upon exposure to the anti-CD3 authenticator. The L234 L235→A234 E235 mutation in the Fc region of the anti-CD3 antibodies provided herein reduces or eliminates cytokine release when the anti-CD3 antibody is exposed to human leukocytes, while the mutations described below maintain significant cytokine release capability. For example, a significant reduction in cytokine release is defined by comparing the level of cytokine release upon exposure to an anti-CD3 antibody having a L234 L235→A234 E235 mutation in the Fc region to the level of cytokine release upon exposure to another anti-CD3-Bobod having one or more of the mutations described below. Other mutations in the Fc region include, for example, L234 L235→A234, A235, L235→E235, N297→A297, and D265→A265.

[0049] The term "cytokine" refers to any human cytokine known in the art that binds to an extracellular receptor expressed on the cell surface and thereby regulates cellular function, including, but not limited to, IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-10, and IL13.

[0050] Pharmaceutical Compositions The anti-CD3 antibodies described herein can be incorporated into pharmaceutical compositions suitable for mucosal administration, e.g., by inhalation, or absorption, e.g., by nasal, intranasal or pulmonary administration.

[0051] For purposes of mucosal therapeutic administration, the active compound (e.g., anti-CD3 antibody) can be incorporated with excipients or carriers suitable for administration by inhalation or absorption (e.g., by nasal spray or nasal drops). For nasal administration, the formulation can be an aerosol in a sealed vial or other appropriate container.

[0052] The pharmaceutical compositions and mucosal (e.g., nasal) dosage forms may further comprise one or more compounds that reduce the rate at which the active ingredient decomposes. Thus, the mucosal dosage forms described herein can be processed into immediate release or sustained release dosage forms. An immediate release dosage form can release the anti-CD3 antibody in a fairly short period of time, e.g., within minutes to hours. A sustained release dosage form can release the anti-CD3 antibody over a period of hours, e.g., up to 24 hours or more, if desired. In either case, delivery can be controlled to be at a substantially predetermined rate over the period of delivery.

[0053] Nasal delivery is considered an attractive route for needle-free systemic drug delivery, especially when rapid absorption and efficacy are desired. Furthermore, nasal delivery may help address issues associated with poor bioavailability, slow absorption, drug degradation, and adverse events (AEs) in the gastrointestinal tract, and may avoid first-pass metabolism in the liver.

[0054] Liquid nasal formulations are primarily aqueous solutions, but suspensions and emulsions can also be delivered. In traditional spray pump systems, antimicrobial preservatives are typically required to maintain microbiological stability in the liquid formulation.

[0055] Metering spray pumps have dominated the intranasal drug delivery market since they were introduced. Pumps typically deliver approximately 25-200 μL per spray, and they offer high reproducibility of the emitted dose and plume shape. Particle size and plume geometry can vary within certain limits and depend on the pump characteristics, formulation, actuator orifice, and applied force. Traditional spray pumps replace the emitted liquid with air, so preservatives are required to prevent contamination.

[0056] Alternative spray systems that avoid the need for preservatives can also be used. These systems use a collapsible bag, a movable piston, or compressed gas to compensate for the amount of liquid dispensed. Solutions with a collapsible bag and a movable piston that compensate for the amount of liquid dispensed offer the added advantage of being able to dispense upside down without the risk of drawing air into the dip tube and compromising the subsequent spray. This may be useful for some products where the patient is bedridden and application is recommended with the head down. Another method used to avoid preservatives is to filter the air that replaces the dispensed liquid through a sterile air filter. In addition, some systems have a ball valve at the tip to prevent contamination of the liquid in the applicator tip.

[0057] The kits described herein can include the anti-CD3 antibody composition as a pre-prepared liquid oral or mucosal dosage form (e.g., nasal) or can include the anti-CD3 antibody composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid oral or mucosal dosage form. When the kit includes the anti-CD3 antibody composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral or nasal administration), the kit can optionally include a reconstitution solvent. In this case, the reconstitution or reconstitution solvent is combined with the active ingredient to provide a liquid oral dosage form of the active ingredient.

[0058] Typically, the active ingredient is soluble in the solvent and forms a solution.The solvent can be, for example, water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component.Suitable non-aqueous components include, but are not limited to, oil; alcohol such as ethanol; glycerin; and glycols such as polyethylene glycol and propylene glycol.In some embodiments, the solvent is phosphate buffered saline (PBS).

[0059] For administration by inhalation, the mucosal anti-CD3 antibody compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant (e.g., a gas such as carbon dioxide) or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0060] In one embodiment, mucosal anti-CD3 antibody compositions are prepared with carriers that will protect the anti-CD3 antibodies against rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. Materials are commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

[0061] Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0062] Dosage, toxicity and therapeutic efficacy of such anti-CD3 antibody formulations can be determined in cell cultures (e.g., cells harvested from animals following mucosal administration of anti-CD3 antibodies) or in experimental animals, e.g., using LD 50 (lethal dose for 50% of the population) and ED 50 The LD (the dose that is therapeutically effective in 50% of the population) can be determined by conventional pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50It can be expressed as a ratio. Compositions that exhibit high therapeutic indices are preferred. Anti-CD3 antibody compositions that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissues to minimize potential damage and thereby reduce side effects.

[0063] Data obtained from cell culture (e.g., cells harvested from animals following mucosal administration of anti-CD3 antibodies) and animal studies can be used to formulate a range of dosages for use in humans. The dosage of anti-CD3 antibodies is preferably administered at an ED that results in little or no toxicity. 50 The dosage is within a range of circulating concentrations including IL-10, TGFβ, or regulatory cells. Doses may vary within this range depending on the dosage form used and the route of administration utilized. For any oral or mucosal anti-CD3 antibody composition used in the methods described herein, the therapeutically effective dose may be estimated initially from cell culture assays (e.g., cells harvested from the animal following mucosal administration of the anti-CD3 antibody). Dosages may be determined based on the IC determined in cell culture to achieve the desired circulating plasma concentration of IL-10 or TGFβ, or regulatory cells. 50 The concentration of the test compound that achieves half-maximal inhibition of symptoms may be formulated in the range including the concentration of the test compound that achieves half-maximal inhibition of symptoms. Such information can be used to more accurately determine useful dosages in humans. Levels of IL-10 or TGFβ in plasma can be measured by methods known in the art, for example, ELISA. Levels of regulatory cells can be measured by methods known in the art, for example, flow cytometry-based methods.

[0064] As defined herein, a therapeutically effective amount (i.e., an effective dose) of an anti-CD3 antibody will depend on the antibody selected, the mode of delivery, and the condition being treated. For example, a single dose may be in the range of about 5-200 μg, about 25-175 μg, about 25-100 μg, about 10-150 μg, about 5-100 μg, about 5-50 μg, about 10-50 μg, about 5-50 μg, about 25-75 μg. In some embodiments, the single dose is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 μg. The daily dose is preferably 50 μg / day. The daily dose can be administered through a single nostril.

[0065] Alternatively, the daily dose may be divided equally between both nostrils.

[0066] As used herein, "dosing regimen" or "dosing regimen" refers to the amount of an agent, e.g., a composition containing an anti-CD3 antibody, administered, and the frequency of administration. The dosing regimen is a function of the disease or condition being treated and can vary accordingly.

[0067] As used herein, the "frequency" of administration refers to the time between successive administrations of a treatment. For example, the frequency can be daily, weekly, or monthly. For example, the frequency can be two or more times per week, three or more times per week, four or more times per week, five or more times per week, six or more times per week, or once a day, etc. The frequency can also be one week, two weeks, three weeks, or four weeks. The particular frequency is a function of the particular disease or condition being treated. In general, the frequency is one or more times per week, and typically three times per week.

[0068] The anti-CD3 antibody compositions can be administered from one or more times per day to one or more times per week (including once every other day). For example, the anti-CD3 antibody compositions are administered once per day every other day for a period of 1, 2, 3, 4 or more weeks.

[0069] As used herein, a "cycle of administration" refers to a repeat schedule of a dosing regimen of administration of an anti-CD3 antibody that is repeated over successive administrations. A cycle can be one week, two weeks, three weeks, or four weeks. For example, an exemplary dosing cycle is a two-week cycle. A subject can receive one to ten cycles of administration. A subject can be considered for one, two, three, four, five or more dosing cycles. Optionally, a drug holiday is provided between dosing cycles. The drug holiday period can be one to four weeks. A drug holiday is preferably one week.

[0070] As used herein, "unit dosage form" or "unit dosage form" refers to physically discrete units suitable for human and animal subjects and packaged individually as known in the art.

[0071] The anti-CD3 antibody compositions can be administered from one or more times per day to one or more times per week (including once every other day). For example, the anti-CD3 antibody compositions are administered once per day every other day for a period of 1, 2, 3, 4 or more weeks.

[0072] Oral or mucosal anti-CD3 antibody compositions can be administered, for example, for about 10-14 days or longer. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or can include a series of treatments.

[0073] The oral or mucosal anti-CD3 antibody compositions may also include one or more therapeutic agents useful for treating autoimmune disorders. Such therapeutic agents include, for example, NSAIDs (including COX-2 inhibitors); other antibodies, such as anti-cytokine antibodies, such as antibodies against IFNα, IFNγ, and / or TNFα; gold-containing compounds; immunosuppressants (e.g., prednisolone and methylprednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporine and tacrolimus; methotrexate; or cotrimoxazole); heat shock proteins (e.g., U.S. Patent U.S. Pat. No. 6,007,821); and treatments for MS, such as beta interferons (e.g., interferon beta-1a, interferon beta-1b), mitoxantrone, or glatiramer acetate.

[0074] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0075] Treatment method Mucosal (e.g., intranasal) anti-CD3 antibody compositions described herein can be administered to a subject to treat or alleviate a sign or symptom of a disorder associated with microglial activation. In some embodiments, mucosal (e.g., intranasal) anti-CD3 antibody compositions described herein can be administered to a subject to prevent a disorder associated with microglial activation. In some embodiments, anti-CD3 is administered in a single dose in the range of doses described herein, e.g., about 5-200 μg, about 25-175 μg, about 25-100 μg, about 10-150 μg, about 5-100 μg, about 5-50 μg, about 10-50 μg, about 5-50 μg, about 25-75 μg. For example, a single dose can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 μg. In some embodiments, the daily dose is 10-200 μg / day. In some embodiments, the daily dose is 50 μg / day. The daily dose can be administered via a single nostril.

[0076] Examples of disorders associated with microglial activation include, for example, neurodegenerative disorders, ischemia-related diseases or injuries, traumatic brain injury, or lysosomal storage diseases.Ischemia-related diseases include, but are not limited to, ischemic creperfusion injury, stroke, and myocardial infarction.Ischemia-reperfusion injury causes damage to lung tissue, heart tissue, or nerve tissue.Traumatic brain injury includes, but is not limited to, repetitive concussion injury or concussion, such as whiplash injury.

[0077] Neurodegenerative diseases include, for example, multiple sclerosis (MS) (e.g., relapsing-remitting MS and secondary progressive MS), Alzheimer's disease (AD), Lewy body disease, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), epilepsy, HIV-associated encephalopathy, and AIDS-associated dementia.

[0078] Mucosal (e.g., intranasal) anti-CD3 antibody compositions described herein can be administered to a subject to treat disorders associated with neuroinflammation. Neuroinflammation is often associated with neurodegenerative diseases, including, for example, AD, PD, MS, and ALS. The level of neuroinflammation can be determined using imaging techniques, such as MRI or PET. In some embodiments, anti-CD3 is administered in a single dose in the range of doses described herein, e.g., about 5-200 μg, about 25-175 μg, about 25-100 μg, about 10-150 μg, about 5-100 μg, about 5-50 μg, about 10-50 μg, about 5-50 μg, about 25-75 μg. For example, a single dose can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 μg. In some embodiments, the daily dose is 10-200 μg / day. In some embodiments, the daily dose is 50 μg / day. The daily dose can be administered via a single nostril.

[0079] In some embodiments, the method results in a reduction in the level of neuroinflammation in the subject by 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%, or at least 95%, compared to the level of neuroinflammation before administration of the anti-CD3 antibody. In some embodiments, the method results in a reduction in neuroinflammation in the subject by 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%, compared to the level of neuroinflammation before administration of the anti-CD3 antibody. Neuroinflammation can be assessed after any suitable treatment period, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the reduction in neuroinflammation persists throughout a washout period (e.g., a washout period of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 12 weeks). Neuroinflammation can be determined, for example, in the whole brain, the cerebral cortex region of the brain, the thalamic region of the brain, the white matter of the brain, and / or the cerebellum region of the brain.

[0080] In some embodiments, a therapeutically effective amount of a mucosal (e.g., intranasal) anti-CD3 antibody composition can be, for example, the amount necessary to reduce microglial activation by at least about 20%. In some embodiments, microglial activation is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% from pre-treatment levels.

[0081] In some embodiments, microglial activation is reduced in the whole brain by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline).

[0082] In some embodiments, microglial activation is reduced in the cerebral cortical regions of the brain by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline).

[0083] In some embodiments, microglial activation is reduced in the thalamic region of the brain by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline).

[0084] In some embodiments, microglial activation is reduced in brain white matter by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline).

[0085] In some embodiments, microglial activation is reduced by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the cerebellar region of the brain.

[0086] The reduction in microglial activation persisted for 1, 2, 3, 4, 5, 6, 7, 8 or more weeks after treatment was stopped.

[0087] The reduction in microglial activation may persist for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more after treatment has stopped.

[0088] In some embodiments, the method results in a reduction in microglial activation in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the level of microglial activation before administration of the anti-CD3 antibody. Microglial activation can be determined by any suitable method known in the art, including, for example, PET scans as described herein. Microglial activation can be assessed after any suitable treatment time, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the reduction in microglial activation persists throughout the washout period (e.g., a 1 week, 2 week, 3 week, 4 week, 5 week, 6 week, 7 week, 8 week, 9 week, or 12 week washout period). Microglial activation can be determined, for example, in the whole brain, the cerebral cortical region of the brain, the thalamic region of the brain, the white matter of the brain, and / or the cerebellar region of the brain.

[0089] Additionally, the concentration of TGF-β1 can be measured. For example, TGF-β1 is measured in peripheral blood using, for example, a cell-based assay, such as enzyme-linked immunosorbent assay (ELISA) or FACS scanning, to monitor the induction of tolerance. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is an amount necessary to increase the level of cells secreting TGF-β1 by about 20% or more. In some embodiments, the level of cells secreting TGF-β1 is increased, e.g., doubled, by at least about 60%, 70%, 80%, 90%, or 100%.

[0090] Additionally, cellular expression of CD74, H2-Ab and / or CX3CR1 can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is an amount necessary to reduce the expression level of CD74 and / or H2-Ab-1 by about 20% or more. In some embodiments, the expression level of CD74 and / or H2-Ab-1 is reduced by at least about 60%, 70%, 80%, 90%, or 100%, e.g., halved.

[0091] In some embodiments, a therapeutically effective amount of a mucosal anti-CD3 antibody composition is an amount necessary to increase the expression level of CX3CR1 by about 20% or more, hi some embodiments, the expression level of CX3CR1 is increased, e.g., doubled, by at least about 60%, 70%, 80%, 90%, or 100%.

[0092] In addition, Ly6C high Cellular expression of CX3CR1 and / or CCR2 on splenocytes can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is high In some embodiments, the expression level of CX3CR1 and / or CCR2 on splenocytes is increased by about 20% or more. high The expression levels of CX3CR1 and / or CCR2 in splenocytes is increased, eg, doubled, by at least about 60%, 70%, 80%, 90%, or 100%.

[0093] In addition, Ly6C high Expression of Dusp1 by splenocytes and Hsp40 can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is administered to a subject at least once a day for at least one week. high In some embodiments, the expression level of Hsp40 in splenocytes is increased by about 20% or more. high The level of expression of Hsp40 of Dusp1 by splenocytes is increased, eg, doubled, by at least about 60%, 70%, 80%, 90% or 100%.

[0094] Methods of treatment or prevention typically involve administering to a subject an oral or mucosal anti-CD3 antibody composition sufficient to stimulate the mucosal immune system. In some embodiments, the methods involve administering an oral or mucosal anti-CD3 antibody composition sufficient to increase IL-10 and / or TGF-β production by T cells, e.g., regulatory T cells, in peripheral blood, e.g., by about 100%, 200%, 300% or more. In some embodiments, the methods involve administering an oral anti-CD3 antibody composition sufficient to reduce T cell proliferation in peripheral blood, e.g., by about 20%; e.g., in some embodiments, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0095] In some embodiments, the method results in at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, or at least a 95% decrease in levels of IL-6, IL-1β, IFN-γ, and / or IL-18 in the subject compared to levels prior to administration of anti-CD3. In some embodiments, the method results in a 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% decrease in levels of IL-6, IL-1β, IFN-γ, and / or IL-18 in the subject compared to levels prior to administration of anti-CD3. The concentration of IL-6, IL-1β, IFN-γ, and / or IL-18 can be determined using any suitable method known in the art or described herein, including, for example, O-link assay. In some embodiments, the concentration of IL-6, IL-1β, IFN-γ, and / or IL-18 is determined in the subject's blood. The levels of IL-6, IL-1β, IFN-γ, and / or IL-18 may be assessed after any suitable time of treatment, for example, after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years. In some embodiments, the reduction in IL-6, IL-1β, IFN-γ, and / or IL-18 levels persists throughout the washout period (e.g., a 1 week, 2 week, 3 week, 4 week, 5 week, 6 week, 7 week, 8 week, 9 week, or 12 week washout period).

[0096] In some embodiments, the method results in an increase in the level of naive CD8 cells in the subject of 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%, or at least 95% compared to levels before administration of the anti-CD3 antibody. In some embodiments, the method results in an increase in the level of naive CD8 cells in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to levels before administration of the anti-CD3 antibody. In some embodiments, the method results in an increase in the level of CD8 naive cells in the subject of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to the level before administration of the anti-CD3 antibody. In some embodiments, the method results in an increase in the level of CD8 naive cells in the subject of 1.5-2-fold, 2-3-fold, 3-4-fold, 4-5-fold, 5-6-fold, 6-7-fold, 7-8-fold, 8-9-fold, or 9-10-fold compared to the level before administration of the anti-CD3 antibody. The level of CD8 naive cells in the subject can be determined using any suitable method known in the art, including, for example, flow cytometry. In some embodiments, the level of CD8 naive cells is determined in the blood of the subject. The levels of naive CD8 cells can be assessed after any suitable time of treatment, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the increase in the levels of naive CD8 cells persists through a washout period (e.g., a 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 12 weeks washout period).

[0097] In some embodiments, the method results in a decrease in the level of CD8 effector cells in the subject of 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%, or at least 95%, compared to the level before administration of the anti-CD3 antibody. In some embodiments, the method results in a decrease in the level of CD8 effector cells in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100%, compared to the level before administration of the anti-CD3 antibody. The level of CD8 effector cells in the subject can be determined using any suitable method known in the art or described herein, including, for example, flow cytometry. In some embodiments, the level of CD8 effector cells is determined in the blood of the subject. The levels of CD8 effector cells can be assessed after any suitable time of treatment, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the decrease in the levels of CD8 effector cells persists throughout a washout period (e.g., a 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 12 weeks washout period).

[0098] In some embodiments, the methods result in an improvement in the Expanded Disability Status Scale (EDSS) score in the subject of 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%, or at least 95%, compared to the EDSS score before administration of the anti-CD3 antibody. In some embodiments, the methods result in an improvement in the Expanded Disability Status Scale (EDSS) score in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the EDSS score before administration of the anti-CD3 antibody. Methods for determining the EDSS score of a subject are described herein and known in the art (see, for example, Kurtske; Neurology. 1983 Nov;33(11):1444-52, which is incorporated herein in its entirety). The EDSS score of a subject can be evaluated after any suitable treatment period, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the improvement in EDSS score persists through a washout period (e.g., a washout period of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 12 weeks).

[0099] In some embodiments, the method results in an improvement in the pyramid score in the subject of 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%, or at least 95% compared to the pyramid score before administration of the anti-CD3 antibody. In some embodiments, the method results in an improvement in the pyramid score in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the pyramid score before administration of the anti-CD3 antibody. Pyramid scores can be assessed after any suitable treatment period, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the improvement in pyramid score persists through a washout period (e.g., a 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 12 weeks washout period).

[0100] In some embodiments, the method results in an improvement in walking ability. Walking ability can be measured, for example, by a 25-foot timed walk test, in which the subject is asked to walk 25 feet as quickly as safely possible. In some embodiments, the method results in an improvement in the time it takes the subject to walk 25 feet by at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 11 seconds, at least 12 seconds, at least 13 seconds, at least 14 seconds, at least 15 seconds, at least 16 seconds, at least 17 seconds, at least 18 seconds, at least 19 seconds, at least 20 seconds, at least 21 seconds, at least 22 seconds, at least 23 seconds, at least 24 seconds, at least 25 seconds, at least 26 seconds, at least 27 seconds, at least 28 seconds, at least 29 seconds, or at least 30 seconds, compared to the time before administration of the anti-CD3 antibody. In some embodiments, the method results in an improvement in the time it takes a subject to walk 25 feet of 1-5 seconds, 5-10 seconds, 10-15 seconds, 1520 seconds, 20-25 seconds, 25-30 seconds, 30-35 seconds, or 35-40 seconds compared to the time before administration of the anti-CD3 antibody. Walking ability can be assessed after any suitable treatment period, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or 5 years after treatment. In some embodiments, the improvement in walking ability persists through a washout period (e.g., a 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 12 week washout period).

[0101] In some embodiments, the method results in stabilization of the subject's EDSS score. In some embodiments, the method results in stabilization of the subject's walking ability. In some embodiments, the method results in stabilization of the subject's microglial activation. In some embodiments, the method results in stabilization of the subject's IL-6, IL-1β, IFN-γ, and / or IL-18 levels. In some embodiments, the method results in stabilization of the subject's levels of CD8 naive cells and / or reduction of CD8 effector cells. "Stabilization" means no substantial increase or decrease (e.g., no more than a 5% increase or decrease) compared to assessment prior to administration of the anti-CD3 antibody.

[0102] In some embodiments, the method includes administering to the subject 8.0 mg / kg of methylprednisolone sodium succinate, e.g., intravenously, e.g., 1-4 hours prior to administration of the mucosal anti-CD3 antibody composition. In some embodiments, the method can include administering to the subject an anti-inflammatory agent, e.g., acetaminophen or an antihistamine, prior to, concurrently with, or after administration of the mucosal anti-CD3 composition.

[0103] In some embodiments, the mucosal anti-CD3 antibody composition is administered simultaneously with one or more second therapeutic modalities, such as symptomatic treatment, high-dose immunosuppressive therapy, and / or autologous peripheral blood stem cell transplantation (HSCT). Such methods are known in the art and may include administration of agents useful for treating autoimmune disorders, such as NSAIDs (including selective COX-2 inhibitors); other antibodies, such as anti-cytokine antibodies, such as antibodies against IFNα, IFNγ, and / or TNFα; gold-containing compounds; heat shock proteins (e.g., as described in U.S. Pat. No. 6,007,821), immunosuppressants (e.g., prednisolone and methylprednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporine and tacrolimus; methotrexate; or cotrimoxazole), and therapeutic cell preparations, such as subject-specific cell therapy, hematopoietic stem cell therapy. In some embodiments, the method includes administering one or more therapies for multiple sclerosis, such as β-interferon (e.g., interferon β1a, interferon β1b), mitoxantrone, or glatiramer acetate. In some embodiments, the method includes administering to the subject one or more non-anti-CD3 immunosuppressants (such as corticosteroids, e.g., prednisolone and methylprednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil; cyclosporine and tacrolimus; methotrexate; or cotrimoxazole), e.g., before, during, or after administration of an oral or mucosal anti-CD3 component. EXAMPLES

[0104] Example 1: Clinical examination and PET imaging of a secondary progressive MS patient treated with intranasal forualumab Patient History The patient is a 61-year-old man with inactive progressive MS on ocrelizumab. He was first diagnosed with MS in March 2001. An MRI of the brain and spine obtained during evaluation of a long-standing worsening of bilateral leg weakness and paresthesia (onset November 2000) showed lesions diagnostically confirmed for MS. He reports a history of neurological symptoms with exertion predating his diagnosis by 20 years. As a high school and collegiate athlete, he noted that strenuous exertion would result in symptoms of left-sided sensory symptoms and horizontal diplopia. After diagnosis, the patient was treated with injectable MS medications, but the patient did not tolerate them and showed insidious clinical progression despite treatment. He was transitioned to Rituxan in 2013 and then to ocrelizumab in November 2018. His MRIs showed no interval changes during this time, but his ambulatory status gradually deteriorated to the point of occasionally requiring a cane by early 2016, then became more stereotyped by November 2017 (EDSS 6, 25-foot walk time approximately 6 seconds), and now wheelchair dependent for distances outside the home, walking fairly slowly with a cane, exacerbating severe imbalance and poor endurance, along with right leg weakness (EDSS 6, 25-foot walk time approximately 20 seconds). Consistent physical therapy was helpful in stabilizing the patient's disease early in the disease course, and adjunctive empirical intravenous steroids have failed to stabilize the patient's disease course. A recent brain and spine MRI performed in May 2021 was unchanged.

[0105] Treatment regimen The patient received intranasal foralumab (anti-CD3) at 50 μg / day (25 μg / nostril × 2 nostrils) 3 times a week (Monday, Wednesday, Friday) for 2 weeks followed by 1 week off for one cycle. This cycle was repeated for a total of 5 cycles, i.e., 15 weeks. The patient walked with a cane and had a baseline EDSS score of 6.0.

[0106] Forualumab was administered intranasally using a controlled particle dispersion device (Kurve Technology) or a standard pipette. Administration was performed at the clinical site and patients were monitored for 1 hour after administration. Forualumab is a fully human IgG1 anti-CD3 monoclonal antibody. The concentration of administered drug was 25 μg / 100 μl. 100 μl was administered into each nostril x 2 nostrils. The total daily dose was 50 μg.

[0107] Neurological evaluation First visit Movement test: Right pronation and fine and pronation movement test

[0108] [Table 1]

[0109] Sensory test: Light touch, pin prick, temperature remains the same. No vibration sensation below the knees.

[0110] Reflexes: [right / left] biceps 3+ / 3+, triceps 2+ / 2+, brachioradialis 3+ / 3+, patella 3+ / 3+, ankle spasm 3+ / 3+, plantar responses upwards bilaterally.

[0111] Coordination / Gait: FNF with moderate left ataxia and severe right ataxia Broad-based, spastic, ataxic gait. Prominent right lower limb spasticity. The Romberg test was positive, and tandem walking could not be tested due to balance issues. -Timed 25 foot walking with a cane in 18.3 seconds. ●EDSS was rated as 6, ambulation index as 4, and disease step as 4.

[0112] Visit at 1.5 months Motor exam: normal bulk and tone; no tremor.

[0113] [Table 2]

[0114] Sensory test: Light and firm, pinprick, temperature unchanged. Vibration sensation decreased below the right knee and slightly decreased in the left toes.

[0115] Reflexes: [right / left] biceps 3+ / 2+, triceps 2+ / 2+, brachioradialis 3+ / 3+, patella 3+ / 3+, ankle spasm 2+ / 2+, plantar responses are extensor bilaterally.

[0116] Coordination / Gait: FNF with bilateral moderate ataxia Broad-based, spastic, ataxic gait Romberg and tandem walking could not be tested. Time walking 25 feet with a cane in 23.26 seconds It didn't stop for the entire 25 foot walk. The EDSS was 6, the ambulation index was 5, and the disease steps were 4.

[0117] Third month visit Motor test: Today's Pronator drift test showed no pronation of the drift.

[0118] [Table 3]

[0119] Sensory test: Lightly touch, prick with a needle, temperature remains the same. There is no vibration sensation below the knee, ankle, or both extremities.

[0120] Reflexes: [right / left] biceps 3+ / 3+, triceps 2+ / 2+, brachioradialis 3+ / 3+, patella 3+ / 3+, ankle spasm 3+ / 3+, plantar responses upwards bilaterally.

[0121] Coordination / Gait: FNF and H2S with moderate bilateral ataxia (more than one on either side) Broad-based, spastic, ataxic gait. Pronounced right lower limb spasticity. The Romberg test was positive and tandem walking could not be tested due to balance. Timed 25 foot walk with a cane: 23.6 seconds, 22.9 seconds EDSS was 6, ambulation index was 5, and disease step was 4. Compared with baseline, at 3 months, there were slight improvements in the distal upper limb muscles, right iliopsoas, and bilateral hamstrings / tibialis anterior. There was no change in walking time or EDSS.

[0122] PET imaging PET radiotracer [F-18]PBR06 [N-(2,5-dimethoxybenzyl)-2-(18)F-fluoro-N-(2-phenoxyphenyl)acetamide] is a second generation PET radioligand targeting the translocation protein of 18 kD (TSPO) overexpressed in activated microglia / macrophages. Strong correlations of [F-18]PBR06 binding with both CD68 expression and TSPO antibody reactivity have been demonstrated in multiple disease models. [F-18]PBR06 has been studied in healthy human volunteers but not in MS, with the exception of a recent study of white and gray matter changes in MS patients.

[0123] Genotyping Before PET scans, blood samples collected at the initial screening visit were genotyped for DNA polymorphisms in the 18-kilodalton translocation protein (TSPO) gene on chromosome 22q13.2 using a TaqMan assay. The index target was a high-affinity binder.

[0124] Radiopharmaceutical production [F-18]PBR06 was manufactured at the hospital's Nuclear Medicine / Biomedical Imaging Research Core Facility following standardized procedures. The product was purified by high performance liquid chromatography and sterilized through a 0.22 μm membrane filter. The final product was dispensed in sterile, pyrogen-free, isotonic solutions for IV administration. Radiochemical purity (RCP) of the radiopharmaceutical was determined using high pressure liquid chromatography. Organic solvents were determined using gas chromatography. The RCP of the radiopharmaceutical was >95%.

[0125] PET acquisition and analysis [F-18]PBR06 was injected into the radial antecubital or other arm or hand veins as a bolus injection for PET scanning using an IV catheter; images were acquired in list-mode acquisition mode using a PET / CT scanner. Standardized uptake value (SUV) images from data obtained between 60 and 90 min after radiotracer injection were reconstructed and interpreted for regional and global radiotracer uptake.

[0126] SUV has been shown to correlate with microglial activation in multiple animal models for PBR06 and other TSPO PET ligands. The SUVR value is the SUV ratio that is further normalized to a "reference" region in the brain. Because a true reference region that is completely devoid of TSPO does not exist in the brain, such reference regions are referred to as "pseudo-reference" regions in the PET literature.

[0127] A first-episode patient with secondary progressive multiple sclerosis (SPMS) (EA1) underwent four [F-18]PBR06-PET / CT scans, the first of which was performed before treatment initiation and subsequent scans after 3 months of treatment with intranasal forualumab, followed by a 7-week washout period and an additional 3 months of treatment with intranasal forualumab (i.e., after 6 months of total treatment with intranasal forualumab).A second SPMS patient (EA2) underwent one [F-18]PBR06-PET / CT scan at baseline and the second of which was performed 3 months after treatment with intranasal forualumab.Interpretation of PET images.

[0128] Medical history A 61-year-old man with SPMS underwent [F-18]PBR06-PET scans before starting treatment with intranasal forualumab, 3 months later, and after 12 weeks of treatment and approximately 7 weeks off drug. PET / CT images were acquired between 60 and 90 minutes after radiotracer injection. Injection doses were 1.55 mCi and 2.45 mCi for baseline and follow-up scans, respectively.

[0129] Pre-treatment / baseline PET scan Coronal, sagittal, and transaxial images of [F-18]PBR06 PET scans revealed widespread, multifocal, increased radiotracer uptake compared with background in the brain parenchyma, with increased radiotracer concentrations particularly prominent in the bilateral thalamus and brain stem. Multiple focal areas of increased radiotracer uptake were also seen in the cortical gray matter and paracortical white matter.

[0130] The mean whole brain SUV was 0.78 g / mL, and the mean thalamic SUV was 0.92 g / mL.

[0131] PET scan 3 months after administration of forualumab Coronal, sagittal and transaxial images of the [F-18]PBR06 PET scan reveal a diffuse decrease in radiotracer uptake in the brain parenchyma compared to the baseline PET scan. Multifocal areas of previously high radiotracer uptake are now significantly less prominent, indicating decreased confluence. Few faint areas of focal increased radiotracer uptake with very mildly increased intensity compared to brain parenchymal background are seen. Bilateral thalamus and brainstem show a significant decrease in PET signal intensity compared to the baseline scan.

[0132] The mean whole brain SUV was 0.59 g / mL and the mean thalamic SUV was 0.70 g / mL, representing a 24.3% and 23.9% decrease compared to pretreatment baseline, which are significantly greater than the test-retest variability seen with [F18]PBR06 and other similar TSPO PET tracers.

[0133] impression There was a significant decrease in global and regional brain [F-18]PBR06 uptake, suggesting reduced glial activation after 3 months of treatment with intranasal foruarumab.

[0134] Holiday PET scan after 7 weeks Coronal, sagittal and transaxial images of the [F-18]PBR06 PET scan reveal a diffuse decrease in radiotracer uptake in the brain parenchyma compared to both the baseline PET scan and the post-treatment scan. Importantly, multifocal areas of previously high radiotracer uptake are now significantly less prominent, indicating decreased confluence. Few faint areas of focal increased radiotracer uptake with very mildly increased intensity compared to brain parenchymal background are seen. Bilateral thalamus and brainstem show a significant decrease in PET signal intensity compared to the baseline scan, with continued decrease compared to the post-treatment scan.

[0135] The mean whole brain SUV was 0.56 g / mL and the mean thalamic SUV was 0.64 g / mL, representing a 27.8% and 31.9% decrease compared to pretreatment baseline, which are significantly greater than the test-retest variability seen with [F18]PBR06 and other similar TSPO PET tracers.

[0136] impression A sustained and persistent decrease in global and regional brain [F18]PBR06 uptake was observed, suggesting a continued decrease in glial activation after 7 weeks of drug withdrawal.

[0137] PET scan 6 months after administration of forualumab The treatment was well tolerated, with no intolerance, side effects, or local irritation observed intranasally throughout the course of treatment. Importantly, PET imaging data demonstrated sustained inhibition of brain inflammation and microglial cell activation, which is associated with cognitive function in MS patients.

[0138] [Table 4]

[0139] Consistent with these clinical and PET observations, treatment downregulated serum levels of proinflammatory cytokines, including interferon-γ (IFN-γ), interleukin (IL)-18, IL-1β, and IL-6, known to be associated with the pathogenesis and progression of multiple sclerosis.

[0140] Clinical disease stabilization Furthermore, clinical disease stabilization was observed as measured by the Expanded Disability Status Scale (EDSS), Timed 25-foot Walk Test (T25FW), 9-Hyper Peg Test (9HPT) and Symbol Digit Modality Test (SDMT).Published PET studies have shown an increase in activated microglial cells in patients with secondary progressive MS (SPMS), which is associated with higher scores on the Expanded Disability Status Scale (EDSS), a scale widely used to measure disability.

[0141] 12 months after forualumab administration In this patient with inactive SPMS treated over 12 months, intranasal forualumab reduced microglial activation on [F-18]PBR06 PET images, reduced levels of proinflammatory cytokines, and demonstrated positive clinical responses without any side effects.

[0142] Example 2: A randomized, double-blind, dose-escalation study of intranasal forualumab in healthy adults method Subjects and study design The study was a randomized, double-blind, dose-escalation study in which subjects received 10 μg, 50 μg, or 250 μg of intranasal forualumab for 5 days at each dose level (n=6) or placebo (n=3). Placebo consisted of phosphate acetate buffer. One spray was administered into each nostril. Two sentinel subjects (one placebo and one active) were allowed at each dose level to evaluate serious adverse events. Each subject participated for 30 days. Participants were healthy volunteers, both male and female, aged 18 to 65 years. All subjects provided informed consent and were treated at the Center for Clinical Investigation (CCI) at Brigham and Women's Hospital. A controlled particle dispersion device from Kurve Technology® was used for intranasal delivery of forualumab. Patients signed an informed consent form. The study was approved by the Mass General Brigham Human Subjects Research Committee (IRB).

[0143] Study Drug Forualumab (28F11-AE; NI-401) is a fully human IgG1 anti-CD3 mAb with a mutated Fc portion that renders it non-FcR binding in vitro and exhibits minimal cytokine release in vivo while maintaining regulation of CD3 / TCR and T cell exhaustion. Forualumab was developed by NovImmune and acquired by Tiziana Life Sciences.

[0144] Clinical and laboratory evaluation Subjects underwent clinical examinations (vital signs) and laboratory tests (hematology, serum chemistry, and urinalysis) on days 7, 15, and 30, and adverse events were noted when blood was drawn for immunological testing. An ENT physical examination, including paranasal endoscopy, was performed by an ENT specialist at the screening visit, visit 5 (last dose day), and visit 9 (day 30). A transnasal questionnaire was administered at all visits during the study period.

[0145] In vitro T cell stimulation Healthy donor PBMCs labeled with cell tracers were stimulated in vitro with soluble anti-CD3 antibodies (UCHT1 or foralumab, 2 μg / ml) and rhIL-2 (5 U / ml) or rhIL2 and anti-CD28 (0.5 μg / ml). After 5 days, cultures were analyzed for viability, CD4 + and CD8 + The specimens were stained for

[0146] Blood sample processing Subjects had blood samples collected at baseline (T1) and at scheduled visits 7 (T2), 14 (T3), and 28 (T4) days after drug administration. Follow-up dates differed slightly; thus, T2 was 7–10 days, T3 was 14–18 days, and T4 was 25–34 days. All blood samples were processed immediately. Plasma was removed by centrifugation of sodium heparin blood collection tubes, after which blood was resuspended and diluted in a 1:1 ratio with PBS and applied to a Ficoll-Hypaque (GE Healthcare) centrifuge to isolate PBMC buffy coats. PBMCs were counted and aliquoted at 2 × 10 7 The cells were resuspended at 10 PBMC / vial and frozen in liquid nitrogen.

[0147] PBMC analysis by flow cytometry PBMCs were thawed in complete RPMI medium (containing 2% human AB serum, Gemini Bio) at 37°C, washed with PBS, and stained for viability (eFluor 506 viability dye, Invitrogen). 5 × 10 6Cells were surface stained for lineage and maturation markers, followed by staining for the intracellular proteins GzmB, Perf, and FoxP3. For surface staining, cells were resuspended in FcR block (30% in MACS buffer, 15 min at 4°C) and then incubated (40 min at 4°C) with a panel of surface antibodies including CD19 (LT19, Miltenyi Biotec), antibodies from Biolegend: CD3 (SK7), CD45RA (HI100), CD127 (A019D5), CD56 (NC1M16.2), CD20 (2H7), and LAPTW4-6H10; antibodies from BD Bioscience: CD4 (SK3), CD8 (SK1), and CD27 (M-T271). After washing with MACS buffer (0.1% FBS / PBS, 4°C), cells were fixed and permeabilized using eBioscience FoxP3 fixation buffer set, incubated in permeabilization buffer containing 10% NRS (normal rat serum, 10 min at 4°C), and then incubated with a series of intracellular antibodies including antibodies from Biolegend: Ki67 (KI67), FoxP3 (206D), IFNγ (4S.B3), IL-17 (BL168), IL-10 (JES3-9D7) and Perf (dG9), and GzmB (GB11, BD Bioscience) (30 min at 4°C). Samples were washed with MACS buffer and each whole sample was analyzed on a BD FACS Symphony flow cytometer with HTS attachment.

[0148] T cell proliferation assay After thawing, 1×10 7PBMCs were stored to generate antigen-presenting cells (APCs) after T cell depletion (CD2 beads, Dynal) and radiation (3200 rad). Total human T cells were isolated from the remaining PBMCs via a Human Negative Pan T Cell Isolation Kit (Miltenyi Biotec) and then labeled with Cell Trace Violet (Invitrogen). Cultures were performed at 5 × 10 cells / ml in RPMI-1640 medium (Life Technologies) supplemented with sodium pyruvate, NEAA, HEPES, glutamine and PennStrep (all from Gibco) and 2% HuS (Gemini Bioproducts) in a minimum of triplicate wells in 96-well U-bottom plates (Costar). 3 Pan T cells / well and 1 x 10 4 T cell / APC cultures were established using 100 APCs. T cell / APC cultures were either unstimulated (PBS) or stimulated with the indicated concentrations of forualumab or commercial Hit3a or UCHT1 anti-CD3 mAbs from BD Bioscience (azide-free / low endotoxin). Some cultures were also supplemented with soluble anti-CD28 (clone 28.2, BD Bioscience, 0.5 μg / ml), rhIL-2 (5 U / ml, Tecileucin**), or TGFβ (Abcam, rhTGFβ, Ab50036). After 5-6 days, cultures were harvested and stained to determine proliferation and expression of cytokines and FoxP3. Cultures were treated with the same PMA / ionomycin and fixation / permeabilization protocol as for the PBMC assay, but stained with the following antibodies: CD4, FoxP3, IFNγ, IL-17, IL-10, TNFα, PD1, PDL1 TIGIT and LAG3 and analyzed on a BD FACS Symphony FACS Analyzer using FlowJo software.

[0149] Single-cell RNA-seq Immune cells from participants who received 50 μg of forualumab were analyzed by scRNA-Seq using the 10x Genomics platform. Specific immune populations (CD4 + T cells, CD8 + T cells, FoxP3 +Cells (Tregs, B cells, monocytes and dendritic cells) were FACS-sorted from T1-T4 PBMCs, hashtagged and combined to generate specific samples. All samples were submitted and processed through the 10x Genomics CellRanger pipeline (v3.0). Analysis of the resulting filtered count matrix was performed using the Seurat single cell toolkit (v4.1) in R. The count matrix was first demultiplexed and filtered to remove doublets and negatives. The demultiplexed samples were then further filtered to remove cells with high mitochondrial gene transcript percentages (>20%), low feature diversity (<1000 UMI) and cells with unusually high transcript counts (>20000). Data were then normalized and scaled using Seurat's default parameters with the NormalizeData, FindVariableFeatures and ScaleData functions. Before clustering the cells, the dimensionality of the dataset was reduced using PCA. Visualization of clustering was completed by using the UMAP algorithm packaged in Seurat. Removal of unwanted effects due to sex differences was completed using the Harmony package (v0.1.0), followed by differential expression analysis performed within Seurat. Accessory packages for analysis and visualization of results were dittoSeq (v1.4.4) and ggplot2 (v3.3.5).

[0150] Antigen Array Antigens were transferred to 384-well polypropylene plates (Genetix, X6004), resuspended in DMSO (1 mg / mL), and spotted onto Epoxy microarray slides (Grace Bio-Labs, 405278) using a microarrayer (Aushon 2470) equipped with solid spotting pins. The microarray slides were then blocked with 1% BSA for 1 h at 37°C and incubated with a 1:10 dilution of samples in blocking buffer for 2 h at 37°C. Slides were then washed and incubated with a 1:100 dilution of goat anti-human IgG Cy3-conjugated and goat anti-human IgM AF647-conjugated detection antibodies (Jackson ImmunoResearch) for 1 h at 37°C. Blocking, probing, and washing steps were performed using a HS 4800 Pro.

[0151] Hybridization station (Tecan) Finally, the slides were scanned using a microarray scanner (Tecan Powerscanner).

[0152] statistical analysis Comparisons of change over time for 57 immunological markers were analyzed separately for each treatment group (patients in the 10 μg, 50 μg, and 250 μg groups, and placebo group combined). Change over time was estimated for each group using a linear mixed-effects model with a fixed categorical effect of time and a random intercept. The categorical effect of time allows for estimation of change from the first measurement to each subsequent measurement. A random intercept was included to account for within-patient correlation. Subjects with missing measurements were included in this analysis.

[0153] result Demographics and Study Summary: Demographic characteristics of each cohort (10 μg, 50 μg, 250 μg, and placebo) are shown in Table 2. Patient disposition is shown in Figure 11. One patient from each treatment cohort discontinued the study for reasons unrelated to the study drug.

[0154] [Table 5]

[0155] safety The drug was well tolerated in all subjects. No systemic effects, including changes in vital signs (temperature, pulse, blood pressure), liver, kidney, or hematological indices (complete blood count with differential count), were observed at any dose during the treatment and follow-up periods. Otorhinolaryngological examinations were unremarkable. No EBV reactivation was observed (Table 3).

[0156] [Table 6]

[0157] In vitro T cell activation with forualumab To determine whether the fully human FcR-modified forualumab antibody induces unbiased human T cell proliferation similar to that induced by other anti-CD3 antibodies commonly used in research, replicate cultures of PBMC stimulated with either forualumab (modified IgG1) or UCHT1 (IgG1) anti-CD3 mAb were established. After 5 days, cultures were stained for viability, CD4 + and CD8 + As shown in Figure 7, the drug inhibited CD8 + Selectively expanded T cells and suppressed CD4 T cell proliferation. Histogram plots (FIG. 7A, bottom) show that the mouse anti-human CD3 mAb, UCHT1, induced greater PBMC proliferation than the fully human anti-human CD3 mAb, forualumab, as indicated by a more extensive cell trace dilution and a lower frequency of non-dividing cells than forualumab-stimulated cultures (FIG. 7A, bottom).

[0158] Since UCHT1 can fully interact with and cross-link FcR and enhance TCR signaling, it is expected that UCHT1 will enhance the proliferation ability. However, CD4 + T cells and CD8 + The culture medium was analyzed to examine the proliferation rate of T cells (Figure 7A, top, Figures 7E and 7F). + T cells and CD8 + The T cell proliferation rate differed markedly depending on the type of anti-CD3 mAb, whereas forualumab inhibited CD8 + T cells were selectively expanded (Figures 7A-7C). + Selective proliferation of T cells occurred in cultures with IL-2 and IL-2 plus anti-CD28, indicating that this was not related to scarcity of costimulation. + The mechanisms underlying preferential activation of T cells remain unclear.

[0159] CD8 + vs. CD4 + Although it may seem unusual to identify an anti-TCR mAb that selectively activates T cells, the humanized, Fc-engineered tepilizumab anti-CD3 mAb also activates CD8 + Tepilizumab has been reported to induce selective proliferation of T cells, and it has been shown to inhibit the proliferation of CD4 T cells in the same culture. + It has been proposed that this arises to induce a population of CD8 FoxP3+ regulatory T cells that kill T cells. Thus, it is possible that forualumab acts via this mechanism to inhibit FoxP3 + regulation CD8 + To test this, we investigated whether forualumab induces CD4 / CD8 T cell proliferation and whether the presence of CD8 T cells is necessary for attenuating CD4 T cell proliferation and whether the CD8 T cells in forualumab-stimulated cultures are required for attenuating CD4 T cell proliferation. + We investigated whether T cells show induction of FoxP3. Thus, negatively isolated CD4 + T cells alone (Figure 7D) or negatively isolated CD4 + and CD8+ "Pan-T" (Figures 7E, 7F) T cell cultures were established that contained both CD4 T cells and T cells. In addition to providing the different soluble anti-CD3 antibodies, the cultures also received irradiated T cell-depleted APCs, and IL-2 (Figures 7D, 7E) or IL-2 / anti-CD28 (Figure 7F). Here, + Compared with cultures with CD8 + In cultures that also included T cells (pan-T cell activation), the frequency of dividing CD4 T cells was significantly reduced (Figure 7D), suggesting that foramumab inhibits CD8 + CD4 via T cells + These results suggest that forualumab induces T cell regulation. However, increased CD8 expression of FoxP3 was observed in forualumab-stimulated cultures.

[0160] The inhibitory effect of anti-CD3 in humans has been proposed to act by altering the balance of Th subsets. Therefore, we next examined whether stimulation with forualumab altered the frequency of Th1 or Th17 (Figure 7G). As shown in Figure 1G, CD8 + In the presence of T cells, CD4 + Expression of IFNγ (Th1), IL-17 (Th17) and TNFα in T cells was significantly increased in pure CD4 + The results were lower than those of T cells stimulated with CD8 + In the absence of T cells, purified CD4 + As a result of stimulating T cells, the expression of immune checkpoint molecules CTLA4 and PDL1 was increased (Fig. 7G). + It was also suggested that CD8 might directly induce inhibitory molecules against T cells. + T cells (CD4 + and CD8 + T cells) responded similarly to forualumab and the UCHT1 anti-CD3 antibody (Figure 7G). These results suggest that forualumab signaling is mediated by regulatory CD8 + This suggests that it may be possible to induce T populations.

[0161] Intranasal forualumab does not modulate CD3 from the T cell surface. IV administration of anti-CD3 mAb induces downregulation of CD3 from the T cell surface. In studies of IV forualumab in Crohn's disease, CD3 modulation was observed at all dose levels (50 μg, 100 μg, 500 μg, and 1000 μg), with the greatest effect seen at doses of 500 μg and 1000 μg. The highest dose of forualumab administered intranasally was 250 μg, which is generally lower than the doses administered IV with forualumab and other mAbs. In animal studies, no downregulation of CD3 on T cells was observed after oral or intranasal administration of anti-CD3, even at doses that result in CD3 modulation administered by the IV route. It is unclear whether lower doses of forualumab and intranasal administration modulate cell surface CD3. To address this, longitudinal PBMC samples from baseline (T1) and from time points T1, T2, and T3 after a 5-day regimen of daily intranasal forualumab were stained and analyzed by cytometry for frequency and mean fluorescence intensity of cells that bound anti-CD3. As shown in Figure 8A, there was no change in frequency of CD3 proliferating cells (top) or intensity of CD3 expression (MFI, bottom) at any dose in samples obtained starting 3 days post-dose (T2). There was also no change in B cells (as a percent of PBMCs) or FoxP3 expression (bottom). + No changes were observed over time in the frequency of Tregs (as a percentage of CD4 T cells) or in the ratio of CD4 to CD8 (not shown).

[0162] The immune effects of intranasal forualumab are manifested at a dose of 50 μg. To determine whether immunological effects are observed after intranasal forualumab administration, PBMCs were stimulated with PMA / ionomycin for 4 hours and stained for surface and intracellular proteins by flow cytometry. Pre-treatment (T1) and post-treatment (T2, T3, and T4) time points were compared between the 10 μg, 50 μg, and 250 μg doses and placebo. A decrease in both proinflammatory activated CD4 and CD8 T cell subsets was observed, mainly observed in the 50 μg dose group. CD27 expression was used instead of CCR7 to define maturation state, as CCR7 expression is decreased on T cells after cryopreservation. CD8 + As shown in Figure 2B, a decrease in the frequency of effector memory cells was observed at T2 and T3. + Further changes observed in cells included CD8 + No changes were observed in central memory cells (Figure 8D), but TEMRA (CD45RA + CD27 - ) frequency, naive cells (CD45RA + CD27 + ) (Figure 8C), and decreased expression of granzyme B. + As for cells, as shown in Figure 2B, at time points 2 and 3, CD4 + As shown in Figure 4, the 5 μg dose increased CD4 + In cells, CD4 + Not only was granzyme B expression reduced in CD4 + Effector Memory (CD3 + CD4 + CD45RA - CD27 - ) and TEMRA (CD3 + CD4 + CD45RA + CD27 - Other changes were observed, including a decrease in the frequency of CD8 + As well as cells, CD4 + No changes were observed in central memory cells. +No changes were observed in Foxp3+ cells. Aliquots of all samples were stimulated with anti-CD3 / IL-2, and at time point 4, there was a change in DN latent binding peptide (LAP)+ cells in patients treated with 50 μg (Figure 8B). No other changes were observed. Of note was a decrease in CD8 perforin (T2 and T4) and an increase in CD8 naive cells (T2) at the 10 μg dose (Figure 8B). At 250 μg, there was an increase in CD4 + There was a decrease in TEMRA and CD4 granzyme B in the 14.1% mice. However, FoxP3 + Decreased expression of TEMRA, granzymes, and perforin was observed with T4 in subjects administered 250 μg, consistent with the observation that immunomodulatory immune effects may be abolished at higher doses.

[0163] [Table 7]

[0164] scRNaseq analysis in 50μg administration Given that immunological effects were primarily observed in subjects receiving the 50 μg dose, scRNaseq was performed on immune populations isolated at baseline and post-dose. To prevent batch effects, cell populations were simultaneously FACS sorted. Consistent with the flow cytometry analysis above, scRNA-seq analysis revealed a reduction in the frequency of CD8 TEMRA and effector memory cells and an increase in the frequency of naive CD8 + The increase in the frequency of T cells was shown (Figure 9). Most of the changes were observed at the first time point, 5 days after administration (T1, baseline vs. T2). FACS-sorted CD8 + , CD4 + We identified differentially expressed (DEG) genes between baseline (T1) and 3–5 days after treatment (T2) in CD8, Treg, and monocyte populations (Figure 9C–E). +T cells showed the highest number of DEGs (109 genes), while CD4 T cells (non-regulatory), Treg, and monocytes showed DEGs with 94, 5, and 3 genes, respectively. Although some of the DEGs functioned in homeostatic cell biological processes (CD8-28%, CD4-53%, Treg-20%, monocyte-33%), most upregulated genes had immunological functions (78 genes in CD8 T cells, 44 genes in CD4 T cells, 4 genes in Treg, and 2 genes in monocytes).

[0165] To elucidate the immune pathways affected by intranasal forualumab, the functions of immune-related DEGs were examined in each cell type. + In T cells (Figure ​(Figure9C), downregulated genes were mainly involved in promoting survival (STAT1, MTRNR2L8, PIM1, FCMR, and IL7R), enhancing cytokine production (BCL11B, ETS1, TRIM22, TDF7, and JUNB), inducing cell dysfunction (TIGIT, CD160, DUSP2), and enhancing cytotoxicity / signaling (PIP4K2A, PIK3R1, XCL1, FLNA, KLRF1, KLRK, and MAPK1). In contrast, upregulated genes in CD8 T cells were anti-inflammatory because they limited protease / proteosome activity (RARRES3, PSMB2), enhanced antioxidant defense (GLRX, IERS), and increased expression of inhibitory receptors (LAIR2, LY6E, and AXNA5), but could also promote migration (CX3CR1 and ITGB1).

[0166] Next, memory CD8 induced by intranasal forualumab + We investigated whether T cell populations contained induction of TIGIT associated with the IV-administered anti-CD3 antibody tepilizumab, which has been shown to be effective in treating T1D patients, and induction of specific KIR family member genes that have recently been shown to play a role in regulating autoimmune responses. Indeed, as shown in Figure 9F, intranasal forualumab-treated CD8 + Effector memory populations had induction of TIGIT (T2) and KIR3DL2 (T2), but not CD8 +The TEMRA population had induction of TIGIT (T3), TGF-B1 (T2, T4) and KIR3DL2 (T2).

[0167] non-regulated CD4 + scRNA-Seq analysis of T cells showed that forualumab treatment downregulated expression in all mature subsets (Figure ​(Figure9D). 9D). The most affected was CD4 + T cells were present in activated subsets (intermediate, memory and LGALS1 signature cells) and showed reduced expression of genes involved in promoting cell migration (CXCR4, NKG7, CCL5, GzmM and SRGN), cytokine production / signaling (ETS1, IL6ST, BCL11B, JUNB, TNFRSF4) and proteosome activation (PCBP2, PSMA6, PSMB10 and PSMA2). In contrast, CD4 + A small number of genes upregulated in T cells appear to function to reduce NF-kB signaling (AES), proteosome activation (RARRES3) and apoptosis (MAL), indicating that they are unlikely to be reactivated. Furthermore, as shown in Figure 9G, memory CD4 + In T cells, intranasal forualumab induced CTLA4 (T2, T3), KLRG1 (T4), and TGFB1 (T2). These results suggest that CD4 + This is consistent with the changes observed when T cells were stimulated with this drug (Figure 7).

[0168] The scRNA-Seq analysis of monocytes (Figure 9E) generated clusters based on genes representative of three classes of monocytes: 1) classical monocytes, associated with antibacterial activity; 2) nonclassical monocytes, involved in immune surveillance; and 3) intermediate monocytes, the most potent inducers of T cell activation. In classical monocytes, DEG genes with decreased expression were either induced by inflammation (LGALS1, SOD2, and CRIP1) or promoting inflammation (GADD45B, DUSP1, FOS, and SRGN). Among the DEG genes in intermediate monocytes, five were genes that affect antigen presentation (HLA-DQB, HLA-DRB, CD74) or decrease monocyte activation status (EFHD2 and RARRES3). In nonclassical monocytes, genes involved in antigen presentation (HLADPA1 and HLA-DPB1) were increased. It has been reported that DQ- and DP-restricted T cells produce higher levels of IL-10, whereas DR-restricted T cells produce higher levels of IFNγ. Thus, intranasal forualumab induces monocytes and promotes less inflammatory immune responses.

[0169] In the Treg population (Figure 12), only four DEGs were identified, all of which had reduced expression: Tregs had reduced expression of JUNB, which may enhance Treg stability by inhibiting Th17 differentiation; USP15, which may reduce sensitivity to TGF-related signaling; and MTRNR2L8, which may alter sensitivity to apoptosis.

[0170] We next examined the relationship of differentially expressed genes identified as having immune functions to determine whether up- or down-regulated genes tended to be associated with pro- or anti-inflammatory responses. + As shown in FIG. 13 for TEMRA cells, 17 / 19 genes promoting inflammatory immune functions were downregulated, whereas 14 / 24 genes suppressing inflammatory immune functions were upregulated.

[0171] Antigen microarray Antigen microarrays are unique tools for the study of the immune system in health and disease. We used an antigen microarray containing a broad panel of antigens (n=550) including self and non-self proteins, heat shock proteins, and infectious agents to study the effect of intranasal forualumab on the immune repertoire. Previously, we used antigen arrays to study immune responses in healthy subjects treated with oral OKT3 antibody. We measured the effect of intranasal forualumab on IgG and IgM reactivity measured at T1 vs. T2, with changes observed primarily in those receiving the 50 μg dose (Figures 10A and 10B). Figure 10A shows that treatment with intranasal forualumab resulted in significant changes in the reactivity of the T cell-dependent IgG repertoire. These findings are consistent with the significant effects on T cell responses detected by functional assays and scRNAseq.

[0172] Example 3: Treatment with intranasal forualumab showed positive clinical data from secondary patients with secondary progressive multiple sclerosis (SPMS) The second patient was treated with intranasal forualumab (anti-CD3) at a dose of 50 μg / day (25 μg / nostril × 2 nostrils). The second patient is a young man in his 40s who was diagnosed with SPMS in 2014 and has had progressive disease since then, resulting in accumulating disability. After completing 3 months of treatment with intranasal forualumab (3 times a week for 2 weeks, followed by 1 week off), the patient showed improvement as measured by microglial activation on PET imaging. The second SPMS patient (Figures 14 and 15) showed an approximately 10-30% decrease in PET signal across brain regions (including cortex, thalamus, white matter, and cerebellum), which is comparable to the PET changes seen after 3 months of treatment in the first SPMS patient (see Table 5) treated with intranasal forualumab. Clinically, the timed 25-foot walk test (Figure 17) and neurological examination (Figure 17) also improved. Both cases 1 and 2 are continuing treatment, 13 and 4 months respectively.

[0173] [Table 8]

[0174] Example 4: Clinical examination and PET imaging of secondary progressive MS patients treated with intranasal foruarumab (latest data) This example describes updated data from the study described in Example 1.

[0175] The first patient had received a total of 14 forualumab treatment cycles to date, with two treatment interruptions of approximately 2 and 3 months, respectively. The patient's EDSS score was stable to improved over the course of forualumab administration, and the pyramidal tract score improved after three cycles of forualumab (Figure 18).

[0176] The patient's timed 25 foot walk (T25FW) went from stable to improved over the course of forualumab administration (Figure 19).

[0177] Microglial activation, as measured by [F-18]PBR06 PET scans, was significantly reduced 3 months after initiation of intranasal forualumab, and this reduction persisted after a 7-week washout and 6 months (Figures 20A and 20B).

[0178] Serum protein measurements of cytokines were performed in batches by Olink assay and showed reduced IL-6, IL-1β, IFN-γ, and IL-18 levels (pg / ml) (Figures 21A-21D, respectively). Cellular immunity studies showed an increase in CD8 naive cells and a decrease in CD8 effector cells and changes in gene expression as measured by single cell RNA sequencing.

[0179] Example 4: Treatment with intranasal forualumab has shown positive clinical data from secondary patients with secondary progressive multiple sclerosis (Spms) (latest data) This example describes updated data from the study described in Example 3.

[0180] The second patient had received a total of 10.5 cycles of forualumab treatment to date, with treatment interrupted for approximately 11 days. The patient showed improvement in his EDSS score on 9 / 12 / 22, after which he demonstrated that he no longer required a cane to walk 100 m, decreasing from 6.0 (walk 100 m with cane) to 5.5 (Figure 22). Pyramid score remained stable (Figure 22).

[0181] The patient showed improvement in his T25FW score on 9 / 12 / 22 (Figure 23). At the two previous visits he required a cane to walk 25 feet. On 9 / 12 / 22 he was able to walk 25 feet without a cane and his walking time was faster (Figure 23).

[0182] Example 5: Intranasal anti-CD3 ameliorates traumatic brain injury by inducing IL-10-dependent Tregs that regulate microglial inflammation introduction Traumatic brain injury (TBI) is a leading cause of death and disability with both direct and indirect costs (Faul et al., Handb Clin Neurol 127, 3-13 (2015)). TBI is associated with long-term morbidity including motor impairment, cognitive decline, and long-term neurodegeneration (Shively et al., Arch Neurol 69, 1245-1251 (2012); Izzy et al. JAMA Netw Open 5, e229478 (2022)). Current treatment focuses on early surgical intervention to limit hematoma expansion and supportive care. However, there are few pharmacological interventions to reduce long-term cognitive sequelae after injury (Langlois, et al, J Head Trauma Rehabil 21, 375-378 (2006); Gordon et al. Am J Phys Med Rehabil 85, 343-382 (2006); McCrory et al. J Athl Train 44, 434-448 (2009); Helmick et al., NeuroRehabilitation 26, 239-255 (2010)). TBI induces a primary mechanical injury followed by secondary biochemical and cellular responses that contribute to neurological damage (Needham et al. J Neuroimmunol 332, 112-125 (2019)). Neuroinflammation is one of the key mechanisms involved in both acute and chronic pathogenesis of TBI (Algattas et al. Int J Mol Sci 15, 309-341 (2013)). TBI activates resident microglia, induces cytokine release, and recruits circulating monocytes and lymphocytes to the CNS, further enhancing inflammation and contributing to secondary injury (Needham et al. J Neuroimmunol 332, 112-125 (2019); Jassam et al., Neuron 95, 1246-1265 (2017)).The exact cellular and molecular mechanisms that lead to neurological deficits after TBI are largely unknown, and there are no therapies targeting this neuroinflammatory process (Jassam et al., Neuron 95, 1246-1265 (2017), Simon et al., Nat Rev Neurol 13, 171-191 (2017)). Thus, there is a great unmet need to identify novel therapies that address chronic CNS inflammation after TBI.

[0183] result Intranasal administration of anti-CD3 mAb improves neuropathological outcomes after TBI To investigate the therapeutic effect of intranasal anti-CD3 in TBI, we used a CCI model of TBI (Smith et al. J Neurotrauma 32, 1725-1735 (2015)), known for its accuracy and reproducibility, to reproduce moderate to severe TBI characteristics, including brain contusion, neuroinflammation, BBB dysfunction, and long-term behavioral outcomes. C57BL6 / J wild-type (WT) mice underwent CCI over the right parietal cortex (tip diameter 1.5 mm, impact depth 1 mm) and were administered either intranasal anti-CD3 (TBI-aCD3) or isotype control (TBI-Iso) starting on the day of injury, once daily for 7 days, and then three times weekly up to one month after injury (Figure 24A). The percentage of ipsilateral and contralateral hemisphere brain edema was assessed 3 days after CCI, and the TBI-aCD3 group showed a significant reduction in ipsilateral hemisphere edema compared to the TBI-Iso control group (Figure 24B). Parenchymal damage volume was examined in the Sham-Iso, TBI-aCD3, and TBI-Iso groups 7 days after injury using 3 Tesla magnetic resonance imaging (MRI) (Figure 24C). There was a significant reduction in ipsilateral lesion volume in the intranasal anti-CD3 treatment group compared to the TBI-Iso control (Figure 24D). Lesion volume was also assessed 1 month after CCI using hematoxylin and eosin (H&E) staining, and there was a significant reduction in ipsilateral lesion volume in TBI-aCD3 mice compared to the TBI-Iso control (Figure 24E).

[0184] Consistent with previous reports, CCI significantly increased monocyte recruitment (CD11b+Ly6c hi ) (Figures 24F and 24I) and a significant increase in microglia / macrophage activation (Iba-1 staining) 1 month after injury compared to Sham-Iso controls (Figure 24G) (Jassam et al. Neuron 95, 1246-1265 (2017); Alam et al. J Neuroinflammation 17, 328 (2020)). CCI also increased cell death as measured by TUNEL staining 7 days after brain injury (Figure 24H). Intranasal anti-CD3 treatment reduced monocyte recruitment, microglia / macrophage activation and cell death after CCI (Figures 24F-24H). Furthermore, intranasal anti-CD3 treatment reduced CD4 expression in both cLNs and brain following CCI (Figure 24I). + FoxP3+ Treg (CD4 + LAP+, but not LAP+), suggesting that anti-CD3-induced Tregs play an important role in suppressing inflammation following CCI.

[0185] Collectively, these findings demonstrate that intranasal anti-CD3 treatment is effective in ameliorating pathological outcomes and treating neuroinflammation and cell death induced in the CCI model of TBI.

[0186] Early intranasal anti-CD3 mAb improves motor and cognitive outcomes in both moderate and severe TBI We next investigated the therapeutic effect of early versus delayed intranasal anti-CD3 treatment on behavioral outcomes after TBI. In the early treatment regimen, intranasal anti-CD3 treatment was administered on the same day of CCI, continued once daily for 7 days, then three times weekly until 1 month after injury. In the delayed treatment regimen, intranasal anti-CD3 was administered on day 14 after CCI, continued once daily for 7 days, then three times weekly until 1 month after injury (Figure 25A). The early treatment regimen improved motor function and coordination as assessed by the rotarod test. Restoration of spatial memory as assessed by the Morris water maze (MWM) and increased time spent in the target quadrant during the probe trial were also observed in the TBI-aCD3 group compared to TBI-Iso controls (Figure 25B). Furthermore, using the open field test, mice treated with intranasal anti-CD3 exhibited less anxiety behavior. No differences were observed in their exploration and activity levels compared to TBI-Iso controls (Figure 25B). However, no improvement was observed in the delayed intranasal anti-CD3 treatment group compared to TBI-Iso controls (Figure 25C).

[0187] To evaluate the impact of intranasal anti-CD3 mAb in more severe forms of TBI, CCI was induced with a 3.0 mm tip diameter and 1.5 mm impact depth over the right parietal cortex, as opposed to the 1.5 mm tip diameter and 1 mm impact depth used in the experiments above. Intranasal anti-CD3 mAb was administered on the same day of CCI, once a day for 7 days, then three times a week for up to one month after injury (Figure 25D). There was improved motor function and coordination in the TBI-aCD3 group compared to TBI-Iso controls (Figure 25E), partial recovery of spatial memory, and increased time spent in the target quadrant during the probe test in intranasal anti-CD3-treated mice compared to TBI-Iso controls (Figure 25E). Furthermore, mice treated with intranasal anti-CD3 exhibited less anxiety behavior compared to TBI-Iso controls, although no differences in exploration and activity levels were observed (Figure 25E).

[0188] Taken together, these data demonstrate that early intranasal anti-CD3 mAb improves behavioral outcomes in both moderate and severe subtypes of TBI.

[0189] Intranasal anti-CD3 mAb modulates chronic microglial inflammatory responses after TBI. Microglia play a key role in neuroinflammation and their activation may contribute to long-term functional deficits after TBI (Jassam et al. Neuron 95, 1246-1265 (2017)). To investigate the effects of TBI and intranasal anti-CD3 treatment on microglial inflammatory transcriptome profiles, microglial single cell suspensions were generated from the ipsilateral hemisphere of mouse brains using a microglia-specific 4D4+ antibody (Krasemann et al. Immunity 47, 566-581 e569 (2017)) at 7 days and 1 month after CCI (Figure 26A). Analysis of the most highly expressed genes in Sham-Iso microglia, TBI-Iso microglia and TBI-aCD3 microglia groups revealed that several microglial genes were most highly expressed, including Cx3cr1, HexB, P2ry12 and Tmem119 (Figure 25F). At 7 days after injury, 927 differentially expressed genes (DEGs) were found in microglia isolated from TBI-Iso vs. Sham-Iso (P<0.05), compared with only 473 DEGs in TBI-anti-CD3 vs. Sham-Iso controls. 695 DEGs were shared between the two comparisons. At 1 month after injury, the number of DEGs in microglia isolated from TBI-Iso vs. Sham-Iso increased to 3954 genes, whereas the number of DEGs in TBI-anti-CD3 vs. Sham-Iso controls decreased to 316 genes. Furthermore, the number of DEGs shared between the comparisons dropped to 488 at 1 month after CCI (Figure 26B). Heatmap signatures of the top 1000 DEGs across the three groups examined revealed that the TBI-Iso and TBI-aCD3 groups had similar microglial transcriptional signatures at day 7, whereas at 1 month post-injury there was a clear modulation of the microglial transcriptional signature in the TBI-aCD3 group towards a Sham-Iso phenotype (Figure 26C).At 7 days after CCI, microglia from the TBI-aCD3 group showed upregulation of genes (Atp2a3, Pik3r5, Atg3) involved in the regulation of microglial activation (Jin et al. Biochem Biophys Res Commun 399, 458-464 (2010); Morales-Ropero et al. Glia 69, 842-857 (2021); Friess et al. Mol Brain 14, 87 (2021); Zhao et al. Neurochem Int 157, 105341 (2022)), as well as upregulation of genes (Pbx2, Nr4a1) involved in neuroinflammatory responses (Wright et al. Genes Immun 9, 419-430 (2008); Rasmussen et al., Mol Brain 10, 43 (2017)), neuronal stress and synaptic dysfunction (Rasmussen et al. Mol Brain 10, 43 (2017); Wang et al. J Neurosci 34, 7253-7265 (2014)), and downregulation of genes involved in regulating cell death (Ccnd2, Sox9, Clu, Nr4a1, Bag6, Bag1, Tgfbr3, Abcb1a, Tgfb2, Atf5, Socs3, Dtnbp1, Birc5, Fmr1, Mical1, Mad2l1, and Rbck1). At 1 month after CCI, microglia from the TBI-aCD3 group had a homeostasis gene signature similar to the Sham-Iso control, including upregulation of genes involved in homeostasis (Tgfbr1, Tgfbr2, Mapk1, App, Hif1a, Smad3, Adgrg1, Mertk, Itgav, Rhob, Atp8a2, Abcc3).In addition, microglia after intranasal anti-CD3 treatment had a less inflammatory signature, similar to sham-iso controls, including downregulation of inflammatory genes such as Tyrobp, Cd36, Cstb, Casp1, Nfkbia, Fcer1g, C5ar1, Psmb3, C1qb and Tlr6 (Kim et al. J Neurosci 28, 4661-4670 (2008); Keren-Shaul et al. Cell 169, 1276-1290 e1217 (2017); Hernandez. et al. Mol Neurodegener 12, 66 (2017). https: / / doi.org:10.1186 / s13024-017-0210-z ).

[0190] The GBP pathway was performed to compare the TBI-Iso and TBI-aCD3 groups to the Sham-Iso control group. TBI-Iso was associated with upregulation of inflammatory biological pathways involved in innate and adaptive immune responses, including IFN-γ, IFN-a, and IFN-b responses, at 7 days and 1 month after injury, consistent with previous reports (Jassam et al., Neuron 95, 1246-1265 (2017)) (Figure 26D, Figure 25C). However, animals receiving TBI-aCD3 showed less upregulation of genes in these proinflammatory pathways and more downregulation of biological pathways involved in phagocytosis, cytokine production, leukocyte activation, and regulation of neuronal maturation compared to TBI-Iso 1 month after injury (Figure 26D).

[0191] Microglia express genes and unique transcriptomic signatures that enable them to perform microglial sensing, homeostatic, and housekeeping functions that are altered by physiological and / or pathological states of the brain (Hickman et al. Nat Neurosci 21, 1359-1369 (2018)). To determine the effects of TBI and intranasal anti-CD3 on these essential microglial functions, microglial cenosome datasets were interrogated for genes and pathways involved in each of these functions (Hickman et al. Nat Neurosci 16, 1896-1905 (2013)).

[0192] Microglia in the intranasal anti-CD3 treated group were associated with upregulation of homeostasis and sensing genes involved in pattern recognition receptors (Tlr1), Fc receptors (Cmtm7), cell-cell interactions (Cd84 and Lag3), and chemoattractant and chemokine receptors (Cx3cr1) at 7 days post-injury compared to TBI-Iso (Figure 26E). Cd33 and Lag3 were the most significantly DEGs in the TBI anti-CD3 treated group compared to TBI-Iso controls at 7 days post-injury. Cd33 activity has been implicated in several processes including receptor and sensor of microglia endogenous ligands, immune cell adhesion processing, and inhibition of cytokine release by monocytes (Crocker et al., Ann NY Acad Sci 1253, 102-111 (2012); Crocker et al., Nat Rev Immunol 7, 255-266 (2007)). Lymphocyte activation gene-3 (Lag3) regulates T cell expansion and limits the duration and intensity of immune responses (Workman et al. Eur J Immunol 33, 970-979 (2003)). Furthermore, TBI anti-CD3-treated groups showed downregulation of key regulators of the microglial inflammatory response to injury, including CD14, at 7 days post-injury (Janova et al. Glia 64, 635-649 (2016)). At 1 month post-injury, intranasal anti-CD3 treatment was associated with upregulation of several TGFβ signaling genes, including Smad3, Tgfbr1, and Tgfbr2, compared to TBI-Iso controls (oller et al., Nat Commun 9, 4011 (2018)). TGF-β is required to maintain the homeostatic state of microglia (Butovsky et al. Nat Neurosci 17, 131-143 (2014)) and to regulate microglia-mediated inflammation after acute brain injury (Taylor et al. J Clin Invest 127, 280-292 (2017)).Furthermore, intranasal anti-CD3 treatment downregulated sensing genes involved in the ECM (Lair1, Ecscr, Itgb2), cytokine receptors (Tnfrsf17), Fc receptors (Fcgr1, Fcgr4, Fcgr3, and Fcer1g), and pattern recognition receptors (Cd74, Tlr6, Selplg) compared to TBI-Iso controls (Figure 26E).

[0193] TBI results in a large amount of myelin and cellular debris, and microglia and macrophages play a key role in debris clearance (Jassam et al., Neuron 95, 1246-1265 (2017)). We analyzed the expression levels of microglial genes involved in phagocytosis and found that TBI was associated with an upregulation of a microglial phagocytic gene signature, including Cybb, C1qa, C1qb, Cyba, Fcer1g, Itgb2, and Tyrobp, especially at 1 month after injury. Conversely, intranasal anti-CD3 treatment was associated with an upregulation of microglial chemotactic and phagocytic transcriptional profiles at 7 days after CCI and a downregulation at 1 month after injury (Figure 26F). Cybb, which encodes the gp91-phox component of the phagocyte oxidase enzyme complex and is involved in the generation of reactive oxygen and superoxide species (Frazao et al. J Cell Biochem 116, 2008-2017 (2015)), was upregulated at day 7 in mice treated with intranasal anti-CD3 mAb but downregulated 1 month after CCI. Other key regulators of microglial phagocytosis, such as Syk and Pik3cg, remained upregulated in the intranasal anti-CD3 group at 1 month after CCI. Intranasal anti-CD3 treatment also upregulated Mertk, a functional regulator of myelin phagocytosis, at 1 month after CCI compared to TBI-Iso controls (Healy et al. J Immunol 196, 3375-3384 (2016)).

[0194] Several studies have reported an association between chronic microglial proinflammatory responses and chronic neurodegeneration after TBI (Jassam et al., Neuron 95, 1246-1265 (2017)). Therefore, we analyzed the expression levels of several proinflammatory microglial genes (Figure 26G), and disease-associated (DAM) (Keren-Shaul et al. Cell 169, 1276-1290 e1217 (2017)) or neurodegenerative (MgnD) (Krasemann et al. Immunity 47, 566-581 e569 (2017)) microglial genes (Figure 26H) after injury. TBI was associated with upregulation of several proinflammatory genes (Ifitm3, Clec7a, Ccl2, Lgals3, IL6, Casp1, CD86, Lyz1, Lyz2, CD40) as well as DAM1 and 2 genes (Tmem119, B2m, Cstb, Cst7, Fth1, Ccl6, Cd9, Cd52, Tyrobp) at 1 month post-CCI compared to Sham-Iso controls. Importantly, these genes were downregulated by intranasal anti-CD3 treatment (Figures 26G-26H). In addition to modulating chronic inflammation, intranasal anti-CD3 upregulated genes involved in synaptic pruning and remodeling, such as Cx3cr1, at 7 days post-injury compared to TBI-Iso controls (Cornell et al. Neural Regen Res 17, 705-716 (2022)) (Figure 26E).

[0195] To assess the phagocytic capacity of microglia after TBI (with or without treatment), we performed in vivo experiments in which TBI-induced lesions were injected with either labeled apoptotic neurons or DPBS on day 6 after injury. In line with the microglial transcriptomic data, anti-CD3-treated animals had a higher microglial phagocytic capacity to engulf apoptotic neurons 16 hours after injection compared to the TBI-Iso group (Figure 26I; Figure 25G-I).

[0196] Consistent with the microglial transcriptome data, RT-qPCR from the ipsilateral hemisphere showed that TBI was associated with increased proinflammatory cytokines (Il12a, Il23, and Ccl5) ​​at 7 days (Il23, Ccl5, IFN-γ, Il6, Il17, Il27, and TNF). Intranasal anti-CD3 treatment increased the anti-inflammatory cytokine Il10 and decreased the expression of several key proinflammatory cytokines (Il6, IFN-γ, TNF, Il17, Ccl5, Il23, and Il12a) at 7 days compared to TBI-Iso controls 1 month after injury (Figure 26J; Figure 26K). Notably, mice treated with intranasal anti-CD3 showed upregulation of brain-derived neurotrophic factor (Bdnf), which has a key role in neuronal survival and is involved in synaptic plasticity, learning and memory, compared to TBI-Iso controls at 1 month post-injury (Figure 26J).

[0197] Taken together, these data indicate that intranasal anti-CD3 modulates post-TBI microglial proinflammatory responses by upregulating microglial homeostasis, sensing, and phagocytosis genes and increasing microglial phagocytosis during the acute phase of injury, and by downregulating proinflammatory and DAM / MgnD microglial genes during the chronic phase of injury.

[0198] Intranasal anti-CD3 mAb ameliorates TBI in an IL-10-dependent manner Nasal anti-CD3 has previously been shown to treat an autoimmune model of progressive multiple sclerosis by inducing IL-10+ Tregs (Mayo et al. Brain 139, 1939-1957 (2016)). In this study, IL-10 expression was increased in the ipsilateral hemisphere of TBI-aCD3 treated animals compared to Sham-Iso and TBI-Iso controls at day 7 after CCI (Figure 26J). There was also upregulation of IL-10-cytokine gene expression in microglia of the TBI-aCD3 group compared to Sham-Iso and TBI-Iso controls at day 7 after CCI (Figure 27A). Thus, we investigated whether IL-10 plays a role in the beneficial effects of nasal anti-CD3 after acute brain injury. Total CD4+ , Treg subtype (CD4 + FoxP3+, CD4 + LAP+, CD4 + FoxP3- and CD4 + The percentage of IL-10-expressing cells in CNS immune cells, including LAP cells, NK cells, microglia, infiltrating monocytes, and neutrophils from the ipsilateral hemisphere of the brain, was examined by flow cytometry at day 7 after TBI in the Sham-Iso, TBI-Iso, and TBI-aCD3 groups. The TBI Iso and TBI-aCD3 groups showed significantly higher levels of total CD4+ / - cells at day 7 after CCI compared to the Sham-Iso controls. + , CD4 + FoxP3+, CD4 + FoxP3-, CD4 + LAP+, CD4 + There was an increase in IL-10 expression in LAP-, microglia, infiltrating monocytes, and NK cells. However, intranasal anti-CD3 treatment increased IL-10 expression in the FoxP3+ Treg population as well as in microglia and NK cells at day 7 after CCI compared to TBI-Iso controls (Figure 27B).

[0199] Next, the effect of blocking the IL-10 receptor on behavioral outcomes after CCI (with and without intranasal anti-CD3 treatment) was examined by intraperitoneal administration of anti-IL10 receptor (aIL-10R) blocking antibodies every 3 days after injury (Figure 27C). The effect of blocking the IL-10 receptor was examined in the Sham-Iso group, the TBI-Iso group, the TBI-aCD3 group, and the TBI-aCD3 group. + The improvements in motor and coordination function, spatial memory, and anxiety-like behavior observed in TBI-aCD3 were abrogated by blocking IL-10 (TBI-aCD3 + aIL10R group) (Figure 27D).

[0200] To investigate the effect of IL-10 receptor blockade on the inflammatory transcriptional profile of microglia, single-cell microglial suspensions were isolated from the ipsilateral hemisphere 1 month after CCI using the microglia-specific 4D4+ antibody (Krasemann et al. Immunity 47, 566-581 e569 (2017)). TBI-aCD3 + aIL10R vs. Sham-Iso shared 1116 DEGs (P<0.05) with TBI-Iso vs. Sham-Iso, whereas TBI-aCD3 vs. Sham-Iso had only 161 DEGs overlapping with TBI-Iso vs. Sham-Iso (Figure 27I). The regulatory effect of intranasal anti-CD3 on microglia (Figures 26C-26D) was abrogated by blocking IL-10, as shown in the heatmap signature of the top 1000 DEGs across groups (Figure 27E; Supplementary Table 1). At 1 month after CCI, TBI-aCD3, similar to TBI-Iso controls, + Microglia from the aIL10R group had a more proinflammatory signature compared to the Sham-Iso and TBI-aCD3 groups (Figure 27E). Next, we performed the GOBP pathway and TBI-aCD3 + We found that the aIL10R group was associated with upregulation of genes involved in proinflammatory pathways, including responses to IFN-γ (Ccl7, Ifitm3, H2-Ab1, Irgm1, Bst2), IFN-a (Ifitm3, Bst2, Ifi204), and IFN-b (Mnda, Ifitm3, Irgm1, Bst2, Ifi204) and complement activation. Similar to TBI-Iso, TBI-aCD3 + The aIL-10R group showed more upregulation in ROS, necrotic cell death and apoptotic pathways compared to the TBI-aCD3 group (FIG. 27F).

[0201] Collectively, these data demonstrate that intranasal anti-CD3-induced CD4+ Tregs modulate microglial responses and improve outcome after injury in an IL-10-dependent manner.

[0202] Tregs suppress microglial activation in vitro To further explore the interaction between Tregs and microglia after TBI, an ex vivo transwell co-culture system was used, in which microglia were isolated from the ipsilateral hemisphere of CCI mice 24 h after injury, and Tregs were isolated from the spleens of a separate cohort of mice subjected to CCI and treated with intranasal anti-CD3 or isotype control for 7 days (Figure 27G). Microglia were placed in the lower chamber and Tregs in the upper chamber, and RT-qPCR of microglia was performed 72 h after incubation. There was an increase in IL-10 expression in TBI-aCD3 microglia, but no difference in the expression levels of other Treg-associated cytokines such as IL4 and Tgfb1 (Figure 27H; Figure 27J). There was also an increase in the expression of the microglial anti-inflammatory marker Cd206 and a decrease in the pro-inflammatory marker Cd14. No differences were found in other proinflammatory cytokines, including IL-6 and TNF, and proinflammatory microglial markers, including Axl, Stat1, Csf1, and Ifitm3, compared with TBI-iso (Fig. 27H; Fig. 27J). These findings indicate that Tregs induced by intranasal administration of anti-CD3 suppress microglial activation in vitro in an IL-10-dependent manner.

[0203] CD4+FoxP3+ regulatory T cells attenuate innate inflammatory responses and improve behavioral outcomes after TBI CD4+FoxP3+ cells were increased in TBI-aCD3 treated animals (Figure 24I and Figure 27B). Therefore, their impact on post-injury behavior and microglial transcriptome profile was evaluated in adoptive transfer experiments. TBI-Iso (Iso-total CD4 + ) and TBI-intranasal anti-CD3-treated mice (aCD3-total CD4 + Total splenic T cells (CD45.2+CD4 + ), and CD45.2+CD4 isolated from anti-CD3-treated animals (aCD3-FoxP3(-)GFP) after CCI. +FoxP3GFP-negative cells were administered intraperitoneally to congenic CD45.1-expressing mice that were untreated but underwent CCI injury. Adoptive transfer was performed at three time points after CCI, with each mouse receiving 2.5 million cells / injection (Figure 28A). Behavior was measured using the rotarod, MWM and open field tests. Total CD4 + In mice receiving T cells, FoxP3 Tregs were depleted in CD4 T cells 1 month after CCI. + Compared to mice receiving T cells, there was improved motor function and coordination, restoration of spatial memory, and increased time spent in the target quadrant during the probe test ( FIG. 28B ). No improvements in anxiety-like behavior or locomotor activity were observed between groups.

[0204] Next, we followed up on the adoptively transferred cells in the recipient mice by transferring cells from CD45.2 mice to CD45.1 mice. Flow cytometric analysis of CD45.2-expressing cells in the recipient brain, cLN, and spleen was performed (Figure 28C). CD45.2-transferred cells were found in all organs analyzed (Figure 28D).

[0205] Total CD4 + and CD4 + To examine the effect of FoxP3(-)GFP-negative cells on the microglial transcriptome profile after injury, we isolated microglia from the ipsilateral hemisphere using a microglia-specific 4D4+ antibody (Krasemann et al. Immunity 47, 566-581 e569 (2017)).

[0206] Iso-total CD4 + , aCD3-total CD4 + Bulk RNA-seq was performed from the aCD3-FoxP3(-)GFP group at 1 month after CCI. 1055 DEGs (P<0.05) were + Vs. Iso-total CD4 + 431 DEGs were found in microglia isolated from aCD3-FoxP3(-)GFP versus Iso-total CD4 +The heatmap signature of the top 1000 DEGs was found to be significantly higher in Iso-total CD4 at 1 month after CCI (Figure 28E). + and aCD3-total CD4 compared with the aCD3-FoxP3(-)GFP group. + We next performed aCD3-total CD4+ / -100% GOBP pathway and compared the groups studied, revealing a clear microglial transcriptome signature (Figure 28F). + We found that the aCD3-total CD4(-)GFP group was associated with a downregulation of several proinflammatory pathways involved in innate and adaptive immune responses, immune effector processes, and antigen presentation, compared with the aCD3-FoxP3(-)GFP group. Furthermore, upregulation of pathways involved in neuronal development and morphogenesis and nerve growth factor receptor signaling was observed in the aCD3-total CD4(-)GFP group. + was observed in treated animals (Figure 28G).

[0207] Consistent with the microglial transcriptome data, RT-qPCR in the ipsilateral hemisphere revealed that Iso-total CD4 + Compared with the aCD3-total CD4 + The group showed increased expression of several anti-inflammatory cytokines (Il10, Il22, and Il2) and growth factors, including Gdnf, at 1 month after CCI (Figure 28H).

[0208] Taken together, these adoptive transfer experiments support the role of CD4 in improving behavioral outcomes and attenuating proinflammatory microglial responses after TBI. + Demonstrating the important role of FoxP3+ Tregs.

[0209] Consideration Neuroinflammation plays a crucial role in both the acute and chronic phases of TBI (Algattas et al. Int J Mol Sci 15, 309-341 (2013)). TBI initiates a complex inflammatory cascade that begins with activation of resident microglia and release of cytokines, followed by recruitment of peripheral monocytes and lymphocytes to the CNS that enhance chronic inflammation and contribute to secondary injury (Needham, EJ et al. J Neuroimmunol 332, 112-125 (2019); Jassam et al. Neuron 95, 1246-1265 (2017)).

[0210] The Treg-dependent immunomodulatory properties of anti-CD3 mAbs in animal models of inflammation and autoimmune disease have been reported previously (Zhang et al. J Immunol 167, 4245-4253 (2001); Sasaki et al. Circulation 120, 1996-2005 (2009); Ochi et al.. Nat Med 12, 627-635 (2006); 408; Ilan, Y. et al. J Clin Immunol 30, 167-177 (2010)) (Mayo et al. Brain 139, 1939-1957 (2016); Herold et al. N Engl J Med 346, 1692-1698 (2002); Mathis et al. Pharmacol Res 120, 252-257 (2017); Notley et al. al. Arthritis Rheum 62, 171-178 (2010)). Intranasal anti-CD3 treatment inhibits IL-10-dependent CD4 +Nasal anti-CD3 ameliorates chronic inflammatory disease through induction of LAP+FoxP3+Tregs, whereas orally administered anti-CD3 induces TGFβ-1 and its downstream signaling (Wu et al. J Immunol 181, 6038-6050 (2008); Mayo et al. Brain 139, 1939-1957 (2016)). The role of intranasal anti-CD3 remains unclear in TBI and other acute brain injury models where the immune system is responding to injury rather than initiating injury. Nasal anti-CD3 mAb induced IL-10+FoxP3+Tregs, attenuated chronic microglial inflammation, reduced peripheral monocyte recruitment, and improved neuropathological and behavioral outcomes after TBI in an IL-10-dependent manner.

[0211] Microglia play a key role in neuroinflammation and their persistent activation contributes to long-term functional deficits and neurodegeneration (Jassam et al. Neuron 95, 1246-1265 (2017)). Time-course changes in microglial transcriptional phenotype with compromised homeostasis, housekeeping and sensing of tissue damage in the early stages after cerebral contusion, as well as recovery, have been previously identified (Izzy et al. Front Cell Neurosci 13, 307 (2019)). In this study, we showed that intranasal anti-CD3-induced FoxP3+Tregs enhanced the homeostatic, sensing and housekeeping microglial phenotype at 7 days after injury, leading to upregulation of genes such as Tlr1, Cmtm7, Cd33, Cx3cr1, Cd84, and Lag3. Moreover, it was associated with an attenuation of the chronic microglial inflammatory transcriptional phenotype after TBI, resulting in downregulation of proinflammatory genes (Ifitm3, Clec7a, Lgals3, Il6, Casp1, Cd86, Lyz1, Lyz2, Cd40). Furthermore, at 1 month after CCI, it downregulated MgnD and DAM genes (Tmem119, B2m, Cstb, Cst7, Fth1, Ccl6, Cd9, Cd52, Tyrobp) associated with neurodegeneration (Krasemann et al. Immunity 47, 566-581 e569 (2017)).

[0212] TBI is associated with neuronal necrosis and death. Microglia play a restorative role by phagocytosing dead or dying cells or debris, participating in synaptic remodeling, and migrating to sites of neuronal death to minimize neuronal damage and restore tissue integrity in the injured brain (Hickman et al., Nat Neurosci 21, 1359-1369 (2018)). Intranasal anti-CD3 was associated with upregulation of genes related to phagocytosis (Cd33 and Cybb) and synaptic pruning and remodeling (Cx3cr1) at 7 days and maintained myelin homeostasis (Tgfbr1, Tgfbr2, Smad3, Mapk1, Hif1a, Adgrg1, Mertk, Itgav, Atp8a2) at 1 month post-injury. Furthermore, it was demonstrated that intranasal anti-CD3 treatment increased microglial phagocytic capacity to engulf apoptotic neurons at 6 days post-injury. They also increased the expression of Bdnf, a key mediator of synaptic plasticity, and increased neuronal TrkB phosphorylation at the injury site (Houlton et al. Front Neurosci 13, 790 (2019)). In removing cellular debris by phagocytosis early after injury and releasing neurotrophic factors and anti-inflammatory cytokines, microglia contribute to the reduced cell death and improved behavioral and neuropathological outcomes observed in the intranasal anti-CD3 group after TBI.

[0213] The role of adaptive immunity after TBI is poorly understood. Several studies have shown T lymphocyte infiltration into the brain after TBI, which plays a role in the neuroinflammatory response after TBI (Xu et al. Cell Prolif 54, e13092 (2021)). Regulatory T cells include FoxP-expanded Tregs and FoxP3 - CD4 including Treg cells +The latter includes a population of T cells, including Th3 and Tr1 cells (Curotto de Lafaille, et al. Immunity 30, 626-635 (2009)). The therapeutic potential of these Tregs in TBI and their regulatory effect on the CNS innate immune system after injury remain largely unknown. Deletion of FoxP3+ Tregs increased CNS infiltration of T cells and expression of inflammatory IFN-γ after TBI. However, the function of Treg-microglia interactions is largely unknown. This study showed that anti-CD3 mAb downregulated microglial activation and induced IL-10-producing FoxP3+ Tregs that migrated to the CNS to improve behavior in an IL-10-dependent manner. Blockade of IL-10 receptors in vivo reversed the therapeutic effect of intranasal anti-CD3 mAb, demonstrating that the Treg / IL-10 axis is a key immunoregulator of innate immune responses and a potential therapeutic target in TBI.

[0214] A major challenge for the treatment of inflammatory diseases is how to induce Tregs in a non-toxic and clinically translatable manner. Nasal anti-CD3 treatment is a unique immunotherapeutic approach that stimulates Tregs to downregulate CNS inflammation. Clinically, nasal anti-CD3 mAb is given immediately to individuals with TBI. Nasal administration of the fully humanized anti-CD3 mAb forualumab reduced lung inflammation and blood inflammatory biomarkers in patients with mild to moderate COVID-19 without side effects (Moreira et al. Front Immunol 12, 709861 (2021)). Of note, in animal studies, nasal anti-CD3 mAb was not detectable in the brain after nasal administration and did not affect the lung's ability to clear bacterial infection (Mayo et al. Brain 139, 1939-1957 (2016)).

[0215] In conclusion, this study identifies a novel therapeutic approach that modulates CNS innate immune responses in an IL-10-dependent Treg manner and is applicable to the treatment of TBI and potentially other types of acute brain injury.

[0216] Materials and Methods Experimental animals Studies were performed using 8-week-old male C57BL6J mice (000664, Jackson Laboratories), B6.SJL-Ptprca Pepcb / BoyJ B6.CD45.1 mice (002014, Jackson Laboratories) and FoxP3-GFP mice (023800, Jackson Laboratory) with littermates. All mice were housed under specific pathogen-free conditions and had food and water available ad libitum. All animals were housed in a temperature- and humidity-controlled room and maintained on a 12 h / 12 ​​h light / dark cycle (lights on at 7:00 AM). Mice were euthanized by CO2 inhalation. All experimental procedures involving animals are in accordance with the Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School and Brigham and Women's Hospital.

[0217] treatment Mice were treated intranasally with a daily dose of 1 μg / day of mouse hamster IgG CD3-specific antibody (clone 145-2C11) or hamster IgG control antibody (BioXCell) dissolved in phosphate-buffered saline (PBS). In some experiments, mice were given 0.5 mg of monoclonal anti-IL-10R blocking antibody (clone 1B1.3A, Bioxcell) intraperitoneally linearly for 7 days at the onset of TBI and then every 3 days for 1 week until the experimental endpoint.

[0218] Controlled Cortical Impact (CCI) The CCI model was used as previously described (Bermpohl et al. J Cereb Blood Flow Metab 27, 1806-1818 (2007)). Mice were anesthetized with 4.5% isoflurane (Anaquest) in 70% nitrous oxide and 30% oxygen using a Fluotec 3 vaporizer (Colonial Medical). Mice were placed in a stereotaxic frame and a 5 mm craniotomy was performed over the right somatosensory cortex using a drill and trephine. The bone flap was removed and discarded, and CCI was induced using a pneumatic cylinder with a 1.5 or 3 mm flat-tipped impounder with a speed of 6 m / s, a depth of 1.0 or 1.5 mm, and a dwell time of 0.8 s (Impact One, Leica Biosystems). The scalp was sutured closed and mice were returned to their cages to recover.

[0219] behavioral research Open Field Test: The open field (OF) test is used to measure general locomotor activity and anxiety-like behavior of animals (Kraeuter et al. Methods Mol Biol 1916, 99-103 (2019)). The OF square chamber is made of blue Plexiglas with dimensions of 30 × 38 × 40 cm. For each test session, animals are allowed to explore the chamber for 15 min. A computer-assisted tracking system and software (Ethovision XT vs.14, Noldus Information Technology) were used to record the animal's behavior during the test period. The total distance traveled (cm) and the % of time spent in the center were measured.

[0220] Rotarod: Rotarod was performed as previously described (Mayo et al. Brain 139, 1939-1957 (2016)). Mice were placed on the rotarod apparatus (Ugo Basile 7650) and accelerated from 4 to 60 RPM for 300 s. Three trials were performed for each animal, and the time it took for the animal to fall and to no longer be able to hold on was recorded and averaged for analysis of motor function.

[0221] Morris Water Maze: The Morris water maze was used to measure spatial learning and memory by training mice to use spatial cues to find a hidden platform to escape the water (Vorhees et al. Nat Protoc 1, 848-858 (2006)). The Morris apparatus is a circular pool with a diameter of 130 cm and a depth of 50 cm. During the first day, the platform was visible and the animals were given three trials to find the platform. During the 4-day training period, the mice received three trials per day to learn how to find the hidden platform. 24 hours after the last training day, a probe trial was performed in which the platform was removed and the mice were allowed to swim for up to 60 seconds. The time the animals spent finding the platform and the time they spent in the target quadrant for the probe trial were calculated by Noldus EthoVision XT tracking software. Heat maps were generated by Ethovision XT software.

[0222] Cerebral edema 72 hours after CCI, the brains were removed and bisected into left and right hemispheres, and each hemisphere was weighed (wet weight). The brains were then dried at 60°C for 48 hours to obtain dry weight. The percentage of brain water content was expressed as (wet-dry weight) / wet weight × 100% (Wu et al. Cell Death Dis 12, 1064 (2021)).

[0223] MRI imaging diagnosis Imaging was performed using a 7.0T Bruker BioSpect® USR. Briefly, mice were gently handled and placed in an isoflurane anesthesia chamber. The mouse was then placed in the imaging device with its nose in front of a tube emitting 2% isoflurane. Electrocardiogram (ECG) leads were placed on the animal's paws and a pneumatic pillow sensor was placed under the abdomen for continuous ECG and respiratory rate monitoring of the anesthetized animal. These waveforms were closely monitored throughout the MRI scan by the MRI operator. The animal was placed on an MRI-compatible bed, which was then placed into the magnet for imaging. The imaging session lasted 15-60 min. The mouse was then returned to its cage and continuously monitored before returning to a fully alert state after being returned to its cage. To generate T2 sequence images, the following parameters were acquired: slice thickness: 0.5 mm, repetition time: 3000 ms, echo time: 50 ms, number of averages: 3, interslice spacing: 0.5 mm, echo train length: 8, acquisition matrix: 200x200, flip angle: 90, field of view: 20 mm. Sequential images were visualized and analyzed using the 3D Slicer platform (Fedorov et al. Magn Reson Imaging 30, 1323-1341 (2012)).

[0224] immunohistochemistry Animals were anesthetized with CO2 until respiratory rate slowed and perfused transcardially with HBSS. Brains were postfixed in 4% paraformaldehyde for 48 h, then transferred to 15% sucrose solution for 24 h, and then finally transferred to 30% sucrose solution for 24 h. Brains were then snap frozen in Tissue-Tek Oct (Sakura, compound 4583) and stored at -80 °C until sectioning. Brains were then sectioned at -20 °C using a cryostat at the Bregma position for each target brain. Sections were cut at 0.2 mm at 4x serial intervals. A total of five sections were placed on Colorfrost PlusTM treated adhesive slides (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20 °C until staining. For immunofluorescence, sections were blocked in 10% normal horse serum solution containing 0.1% Triton X-100, 1% glycine, and 2% bovine serum albumin. Slides were incubated with anti-Iba1 (rabbit, 1:1000, Wako) overnight at 4°C. The next day, sections were washed and incubated with AlexaFluor 647 goat anti-rabbit IgG (1:1000, Abcam, ab150075) for 1 h at room temperature. Sections were also stained with hematoxylin and eosin (HE; Abcam, ab245880) and TUNEL (TUNEL Assay BrdU-Red, Abcam, ab66110) according to the corresponding kit protocols. Iba-1 and TUNEL stained slides were co-stained with DAPI mounting medium (Vector Laboratories, UX-93952-24). Five animals were used per group for each staining. Images were taken with a Leica DMi8 Widefield Microscope at a 20x objective.

[0225] Image analysis Analysis of the percentage of Iba-1 and the number of TUNEL-positive cells per surface area was performed on five micrographs per animal (n=4 or 5). Analyzed sections were taken between 300 and 1500 micrometers laterally from the coronal plane. Images of the brain injury area were generated using each scanned micrograph. All images were analyzed using ImageJ software (National Institutes of Health, https: / / imagej.nih.gov / ij / ). Images were split by color channel, channels of interest were thresholded using the Yen settings, and the percent area and number of positive cells were quantified as previously described (Izzy et al. Int J Mol Sci 22 (2021)).

[0226] Sorting of microglia by flow cytometry For microglial cell sorting, mice were anesthetized with CO2 until respiratory rate slowed, then perfused transcardially with 50 mL of Hank's balanced salt solution (HBSS) containing heparin (1:1000). After perfusion, the ipsilateral hemisphere was homogenized using a Dounce glass tissue homogenizer. Cells were separated by Percoll (GE Healthcare Life Sciences) 30% gradient centrifugation. Cells were isolated from the Percoll layer and stained for 30 min on ice in blocking buffer containing 0.2% bovine serum albumin (BSA, Sigma-Aldrich) in HBSS with a combination of PE / Cy7 rat anti-mouse CD11b (Biolegend, #101216, 1:100), APC / Cy7 rat anti-mouse CD45 (Biolegend, #103116, 1:100), FITC rat anti-mouse Ly6C (Biolegend, #128006, 1:200) and APC rat anti-mouse 4D4 (Krasemann et al. Immunity 47, 566-581 e569 (2017)) (marking resident microglia; 1:1000). Cell sorting was performed using a FACSAriaIII cell sorter (Becton Dickson). Microglial cells were identified as CD45+CD11b+Ly6C-4D4+, and dead cells were also excluded based on 7-AAD (BD Bioscience) staining. Cells were sorted directly into 1.5 mL Eppendorf tubes and stored at -80°C.

[0227] Intracellular staining by flow cytometry Intracellular cytokine staining and cell isolation were performed as previously described (Rezende et al. Nat Commun 9, 3151 (2018)). Ipsilateral brain hemispheres were isolated using a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec #130-092-628) according to the manufacturer's instructions. After enzymatic dissociation, cells were dissociated using Percoll (GE Healthcare Life Sciences) as described above. Cells isolated from brain were incubated for only 2 hours instead of 4 hours for both spleen and cLN cells. Flow cytometry collection was performed on a Fortessa or Symphony (BD Biosciences) by using DIVA software (BD Biosciences) and data were analyzed with FlowJo software version 9.9 or 10.1 (TreeStar Inc.). Dead cells were excluded using intracellular staining antibodies using the Zombie Aqua Fixable Viability Kit (Biolegend, #423102, 1:1000) or Zombie UV (Biolegend, #423108, 1:1000).The staining antibodies used were AF700 anti-CD45 (Biolegend, #103128, 1:200), BV785 anti-CD11b (BD Biosciences, #740861, 1:200), BV605 anti-CD3ε (Biolegend, #100351, 1:100), PE / Cyanine7 anti-TCR-β (Biolegend, #109222, 1:100, BUV661 anti-CD45 (BD Biosciences, #565079, 1:200), PE anti-CD4 (BD Biosciences, #553730, 1:100), FITC anti-FoxP3 (eBioscience, #11-5773-82, 1:100), PE anti-LAP (Biolegend, #141404, 1:100), PE / Dazzle 594 anti-IL10 (Biolegend, #505034, 1:100), BV570 anti-Ly6G (Biolegend, #127639, 1:100), BV605 anti-Ly6G (Biolegend, #127639, 1:100), APC anti-FCRLS (1:1000) provided by Dr. Butovsky, BUV395 anti-NK1.1 (BD Biosciences, #564144, 1:100), and AF700 anti-Ly6C (Biolegend, #128024, 1:200).

[0228] Quantitative polymerase chain reaction RNA was extracted with RNeasy® columns (Qiagen), cDNA was prepared and used for quantitative PCR (Applied Biosystems™, 437466) and results were normalized to Gapdh (Mm99999915 g1). AppliedBiosystems, IL10 (Mm01288386 m1), IL6(Mm00446190 m1), Tnf(Mm00443258 m1), IL-1b (Mm00434228 m1), IL-2 (Mm00434256 m1), IL-23a (Mm00518984 m1), IL-18 (Mm00434226 m1), INFg(Mm01168134 m1), Gapdh(Mm00484668 m1), IL-3 (Mm00439631 m1), IL-27(Mm00461162 m1), Tgfa(Mm00446232 m1), IL18(Mm00434226 m1), IL12a (Mm00434169 m1), Bdnf(Mm04230607 s1), Gdnf(Mm00599849 m1), CCL5(Mm01302427 m1), Csf1(Mm00432686 m1), Lgals3(Mm00802901 m1), Ifitm3(Mm00847057 s1), Stat1(Mm01257286 m1), Axl(Mm01169744 m1), CD14(Mm01158466 g1), Mrc1(CD206)(Mn01329359 m1), IL4(Mn00445259 m1), Tgfb1 (Mm01178820 ml), IL-17a (Mn00439618 m1), IL-21 (Mm00517640 m1). The expression ratios of each gene were calculated using the 2-ΔΔCt method.

[0229] Isolation of primary neurons Primary neuron isolation was performed as previously described (Krasemann et al. Immunity 47, 566-581 e569 (2017)). Briefly, primary neurons were prepared from E18 embryos. Cell density was measured using a hemocytometer and cells were seeded. DMEM containing 10% FBS was used for initial plating and after 3 hours the medium was replaced with Neurobasal supplemented with 1x B27 (Invitrogen). The medium was changed every 3 days.

[0230] Induction of apoptosis and labeling of neurons Neuronal apoptosis and labeling were performed as previously described (Krasemann et al. Immunity 47, 566-581 e569 (2017)). Neurons were irradiated with UV light (302 nm) at an intensity of 6 × 15 W for 15 min. Apoptotic neurons were labeled with a labeling dye (Alexa488 5-SDP Ester or Alexa405 NHS Ester, Life Technologies / Thermo Fisher Scientific). Neurons were resuspended at a density of 260,000 cells per 4 uL for stereotactic injection.

[0231] Stereotactic injection Mice were anesthetized by intraperitoneal injection of ketamine (100mg / kg). Apoptotic neurons or Sterile DPBS were injected into the lesion of TBI mice at two depths, 1mm and 2mm. 2uL was injected at each depth using a stereotaxic device (Harvard Apparatus). After recovery from surgery, animals were returned to their cages. After surgery (16 hours), animals were euthanized by CO2 inhalation and flow cytometry analysis of phagocytic microglia was performed.

[0232] Adoptive transfer To test the in vivo regulatory function of intranasally derived T cells, freshly isolated whole splenic CD4 T cells were depleted of FoxP3+ cells from anti-CD3 or isotype control treated TBI mice (CD45.2) during the acute phase of TBI (day 7). + or CD4 + T cells were transferred into a new cohort of TBI (CD45.1) mice at the onset of TBI, on days 14, and 30. Each recipient received 2.5 × 10 6T cells were injected intravenously. Prior to sorting, splenocytes were purified and enriched on a magnetic MACS separator using a CD4 cell isolation microbead kit (Militenyi Biotech, #130-104-454). Cell sorting was performed using a FACSAriaIII cell sorter (Becton Dickson) and APC anti-mouse CD4 antibody (GK1.5, Biolegend), and live CD4 was identified using 7-AAD (BD Bioscience). + Identify the population and use the FITC channel to identify FoxP3+ depleted CD4 + The FoxP3+ population in the population was excluded.

[0233] In vitro cell culture Sorted 4D4+ microglia 24 hours after TBI were cultured at 200,000 cells in 24-well plates (Kemtec™ 4422A) as previously described (Xie et al. Eur J Immunol 45, 180-191 (2015)). Microglia culture medium consisted of 10% fetal bovine serum (FBS; Gibco, #10438026), 100 U / mL penicillin-streptomycin mixture (Lonza, #DE17-602E), supplemented with Dulbecco's modified Eagle's medium (DMEM) / F-12 Glutamax medium (Gibco, #10565018). Total CD4 +For Tregs, anti-CD3 and isotype control treated TBI mice were cultured in a 10% Fibroblast Growth Hormone (FBS) medium consisting of 10% fetal bovine serum (FBS; Gibco, #10438026), 100 U / mL penicillin-streptomycin mixture (Lonza, #DE17-602E), 55 μM 2-mercaptoethanol (Gibco, #21985023), 1% sodium pyruvate (Lonza, #BE13-115E) and 1% HEPES (Lonza, #BE17-737E) at Roswell Park Memorial Hospital. Cells were sorted into lymphocyte culture medium supplemented with Recombinant Protein Institute (RPMI) 1640 medium (Gibco, #11875119) and placed on top of hanging cell culture 0.4 μm inserts (Millicell, PTHT24H48) at 800,000 cells per insert, cultured microglia, and the assay was left for 72 hours in a CO2 cell culture incubator (InCusafe). After 72 hours, microglia were lysed with Buffer RLT, RNA was extracted with RNeasy® columns (Qiagen), and qPCR was performed.

[0234] Bioinformatics: Microglial bulk RNA sequencing: Bulk RNA sequencing was performed as previously described (Butovsky et al. Nat Neurosci 17, 131-143 (2014)). Briefly, 2000 isolated microglia CD45+CD11b+Ly6C-4D4+ were lysed in 5 μl TCL buffer + 1% β-mercaptoethanol. Smart-Seq2 libraries were prepared and sequenced by the Broad Genomic Platform. cDNA libraries were generated from sorted cells using Smart-seq2 protocol 5. RNA sequencing was performed using an Illumina NextSeq500 with the High Output v2 kit, generating 2 × 38 bp reads. Processing of bulk RNA-seq data was based on an established computational pipeline (Pertea et al. Nat Protoc 11, 1650-1667 (2016)). Sequencing data were demultiplexed and provided by the Broad Institute in FASTQ format. Sequencing quality control was assessed using FastQC. Trimmomatic was used for adapter trimming of reads. Reads were then aligned to the “mm10” reference genome using HISAT. Generated SAM files were converted to BAM files using SAMtools. StringTie was used for transcript assembly and quantification. Transcript abundances were then imported into R Studio (version 4.1.2) and converted to gene-level estimated counts using Bioconductor’s “tximport” package (version 1.22.0). Genes that achieved less than 10 counts summed across all samples were considered very low expressed genes and therefore excluded. Sample read counts were normalized using the variance-stabilized transformation method (VST) from the DESeq2 (version 1.34.0) built-in VST function.These normalized sample read counts were used to plot heatmaps using pheatmap (version 1.0.12) and ComplexHeatmap (version 2.13.1) and barplots using ggpubr (version 0.4.0) and ggplot2 (version 3.3.6). Principal component analysis (PCA) plots were generated utilizing the DESeq2 built-in PCA function using default settings.

[0235] There were three different cohorts that underwent separate RNA sequencing: intranasal anti-CD3 cohort, intranasal anti-CD3 / anti-IL10R cohort, and adoptive transfer cohort. For the intranasal anti-CD3 cohort, there were two time points: 7 days and 1 month after TBI. Samples were divided into three different groups for each time point separately: Sham-Iso, TBI-Iso, and TBI-aCD3. For the intranasal anti-CD3 / anti-IL10R cohort, samples were divided into three different groups at 1 month after TBI: Sham-Iso, TBI-Iso, and TBI-aCD3+aIL10R. For the adoptive transfer cohort, samples were divided into three different groups at 1 month after TBI: Iso-total CD4+, aCD3-total CD4+, and aCD3-FoxP3(-)GFP. To directly compare differences in expression between TBI-aCD3 and TBI-aCD3+aIL10R versus TBI-Iso and Sham-Iso, the intranasal anti-CD3 / anti-IL10R cohort was combined with the 1-month post-TBI group of the anti-CD3 cohort and batch effects were corrected using ComBat-seq 91 via the Sva package (version 3.42.0).

[0236] Differential gene expression and pathway analysis: Differential gene expression analysis was performed with DESeq2. Genes identified using DESeq2 characterized by a P-value <0.05 (Benjamini-Hochberg method) were considered as significant differentially expressed genes (DEGs). Comparison of gene expression across three or more sample groups was performed using the reduced likelihood ratio test (LRT) method. For pairwise comparison of gene expression between two different sample groups and pathway analysis, Wald Test was used with standard parameters and log2 fold changes were subsequently reduced using DESeq2. Pairwise comparison of differentially expressed genes was visualized using DiVenn. All gene set and pathway analyses were performed via the GAGE ​​package (version 2.44.0). Statistical significance for all path analyses and tests was defined as a P-value <0.05. Statistical analysis: Statistical analysis was performed using GraphPad Prism 9 software. Data are presented as mean ± sem and statistical significance between groups was assessed using Student's t test (unpaired) or one-way and two-way ANOVA with Tukey's multiple comparison test. All n and P values ​​as well as statistical tests are shown in the figure legends.

[0237] References [ka]

[0238] Other embodiments Although the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative, not limiting, of the scope of the invention, which is defined by the appended claims. Other embodiments, advantages, and modifications are within the scope of the claims.

Claims

1. 1. Use of an anti-CD3 antibody for treating a neurodegenerative disorder, an ischemia-related disease or injury, a traumatic brain injury, or a lysosomal storage disease in a subject, the use comprising intranasally administering to the subject a daily dose of about 10 μg to 200 μg of the anti-CD3 antibody.

2. 2. The use according to claim 1, wherein the neurodegenerative disease is multiple sclerosis (MS), Alzheimer's disease (AD), Lewy body disease, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), epilepsy, HIV-associated encephalopathy and AIDS-associated dementia.

3. The use according to claim 1, wherein the ischemia-related disease is ischemia-reperfusion injury, stroke, or myocardial infarction.

4. The use according to claim 3, wherein the ischemia-reperfusion injury is in lung tissue, cardiac tissue, or nervous tissue.

5. 2. The use according to claim 1, wherein the traumatic brain injury is a concussion or whiplash injury.

6. 6. The use according to claim 5, wherein the concussion is a repetitive concussion injury.

7. The use according to claim 1, wherein the lysosomal storage disease is Niemann-Pick disease.

8. The use according to any one of claims 1 to 7, wherein the sign or symptom of a disease associated with microglial activation is amyloid plaque formation.

9. The use according to any one of claims 1 to 8, wherein the anti-CD3 antibody is a monoclonal or polyclonal antibody.

10. The use according to any one of claims 1 to 9, wherein the anti-CD3 antibody is fully human, humanized or chimeric.

11. 11. The use according to any one of claims 1 to 10, wherein the anti-CD3 antibody comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 5), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 6), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 7).

12. The use according to any one of claims 1 to 11, wherein the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO:8 and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO:

9.

13. The use according to any one of claims 1 to 12, wherein the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO:

11.

14. The use according to any one of claims 1 to 13, wherein the daily dose is administered once a day.

15. 15. The use according to claim 14, wherein the daily dose is 50 μg.

16. The use according to any one of claims 1 to 15, wherein the daily dose is divided equally between each nostril.

17. 17. The use according to claim 16, wherein the daily dose is administered three times a week.

18. 18. The use of any one of claims 1 to 17, wherein the daily dose is administered to the subject for at least one cycle, the cycle being once daily, three times a week for two weeks.

19. 19. The use according to claim 18, wherein the cycle is repeated 2 to 10 times.

20. 20. The use according to claim 18 or 19, wherein the cycle is followed by a drug holiday.

21. 21. The use according to claim 20, wherein the drug holiday is one week.

22. 22. The use of any one of claims 1 to 21, wherein the method results in an improvement in the EDSS score in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the EDSS score before administration of the anti-CD3 antibody.

23. 23. The use of any one of claims 1 to 22, wherein the method results in an improvement in pyramidal tract score in at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of subjects compared to the pyramidal score before administration of the anti-CD3 antibody.

24. 24. The use of any one of claims 1 to 23, wherein the method results in an improvement in walking ability as measured by a 25-foot timed walk test in the subject of at least 2 seconds, at least 3 seconds, at least 5 seconds, at least 10 seconds, at least 15 seconds, or at least 20 seconds compared to walking ability before administration of the anti-CD3 antibody.

25. 25. The use of any one of claims 1 to 24, wherein the method results in a reduction in microglial activation as measured by PET scan in the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the level of microglial activation before administration of the anti-CD3 antibody.

26. 26. The use of any one of claims 1 to 25, wherein the method results in a decrease in the levels of IL-6, IL-1β, IFN-γ, and / or IL-18 in the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the levels before administration of the anti-CD3 antibody.

27. 27. The use of any one of claims 1 to 26, wherein the method results in an increase in the level of CD8 naive cells and / or a decrease in CD8 effector cells in the subject by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to the levels before administration of the anti-CD3 antibody.

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