Compositions and methods for treating neurological disorders

Abstract

The present invention relates to artificial, brain-penetrating therapeutic compounds and methods of use thereof. A method for intranasal administration of an active agent is also disclosed. TIFF2025508682000073.tif38128

Description

[Technical field]

[0001] Related Applications This application claims priority to or the benefit of U.S. Provisional Application No. 63 / 306,985, filed under 35 USC 119, filed February 4, 2022, No. 63 / 347,113, filed May 31, 2022, and No. 63 / 480,398, filed January 18, 2023, each of which is incorporated by reference in its entirety.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety, and the disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights whatsoever.

[0004] Government Interests This invention was made with Government support under Grant No. R01 048125 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0005] FIELD OF THEINVENTION The present invention relates to artificial, brain-penetrant therapeutic compounds and methods of use thereof. [Background technology]

[0006] 2. Background of the Invention Alzheimer's disease (AD) is a progressive neurological disorder that causes the brain to shrink and brain cells to die. AD is the most common cause of dementia, which is a continuing decline in thinking, behavioral, and social skills that affects a person's ability to function independently. Worldwide, approximately 55 million people currently have dementia, and this number is expected to increase to 78 million in 2030 and 139 million in 2050. There are no effective treatments that cure AD or alter the disease process in the brain. Summary of the Invention

[0007] In general, the present disclosure relates to compounds that can cross the blood-brain barrier (BBB) ​​of a patient. The compounds may include a "payload" or active agent, which may be a therapeutic agent, a cytotoxic agent, an imaging agent, etc. The compounds generally include a moiety for delivery of the active agent across the BBB. In embodiments, the moiety may include one or a combination of a carrier agent, a bridging moiety (e.g., an amino acid bridging moiety), and a linker (e.g., a flexible linker).

[0008] One aspect of the present invention relates to a therapeutic compound that can cross the blood-brain barrier of a patient. In an embodiment, the therapeutic compound comprises a therapeutic agent conjugated to a positively charged amino acid bridge, a flexible linker, a carrier agent, or a combination thereof. In an embodiment, the therapeutic compound comprises a therapeutic agent conjugated to a positively charged amino acid bridge, a flexible linker, and a carrier agent. The carrier agent can be configured to cross the blood-brain barrier. The amino acid bridge can be configured to cross the blood-brain barrier.

[0009] Another aspect of the invention includes a moiety for delivery of a therapeutic agent across the blood-brain barrier of a patient. In certain embodiments, the moiety includes a positively charged amino acid bridge, a flexible linker, and a carrier agent. In embodiments, the carrier agent is configured to cross the blood-brain barrier via receptor-mediated transcytosis (RDT). In embodiments, the amino acid bridge is configured to cross the blood-brain barrier via adsorptive-mediated transcytosis (AMT).

[0010] In various aspects and embodiments disclosed herein, the positively charged amino acid comprises lysine, arginine, histidine, or a combination thereof. The positively charged amino acid may comprise two consecutive lysine residues.

[0011] In various aspects and embodiments disclosed herein, the flexible linker comprises 6-aminohexanoic acid (Ahx).

[0012] In certain aspects and embodiments disclosed herein, the carrier agent comprises Angiopep-2.

[0013] The therapeutic agent can include an anti-osteopontin (OPN) antibody. The antibody can be conjugated to a moiety for delivery of the therapeutic agent across the blood-brain barrier. In some embodiments, the antibody can be conjugated to the moiety via MFCO-N-hydroxysuccinimide ester.

[0014] Further aspects include pharmaceutical compositions for treating a neurodegenerative disease in a patient, comprising an anti-osteopontin (OPN) antibody. In embodiments, the pharmaceutical composition comprises a means for crossing the blood-brain barrier. In embodiments, the neurodegenerative disease includes Alzheimer's disease, multiple sclerosis, Parkinson's disease, limb atrophic lateral sclerosis, and the like. In certain embodiments, the means for crossing the blood-brain barrier comprises any of the various subembodiments described herein.

[0015] In another aspect, the invention includes a method of delivering an agent to the central nervous system of a patient in need thereof. In some embodiments, the method includes peripherally administering an agent (e.g., a therapeutic agent or therapeutic moiety described herein) and allowing the agent to cross the blood-brain barrier. In some embodiments, the agent can be administered intravenously. In some embodiments, the agent can be delivered to the brain. In some embodiments, the active agent can be an antibody.

[0016] In some embodiments, an active agent (e.g., a therapeutic agent) can be administered using a nasal or intranasal route to deliver the active agent to the brain. In some embodiments, an active agent delivered intranasally may not include a moiety for delivery of the agent across the blood-brain barrier. In some embodiments, intranasal delivery of an active agent may result in delivery to the brain without crossing the blood-brain barrier. In some embodiments, an active agent delivered intranasally may result in olfactory transmission of the active agent to the brain. In some embodiments, the active agent may be an antibody.

[0017] Yet another aspect includes a method of treating a neurodegenerative disease in a patient. In an embodiment, the method includes administering to the patient any of the pharmaceutical compositions disclosed herein. The neurodegenerative disease may include Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and the like. In an embodiment, the means for crossing the blood-brain barrier includes any of the various subembodiments described herein.

[0018] In certain embodiments, the therapeutic compound comprises the structure of FIG.

[0019] Other objects and advantages of the present invention will become readily apparent from the ensuing description.

[0020] Other objects and advantages of the present invention will become readily apparent from the ensuing description. [Brief description of the drawings]

[0021] [Figure 1]Figure 1A-F shows that CD11c+ microglia differentiate from CD11c- precursors during phagocytosis of apoptotic neurons (AN) early during development independent of microglial activation. (A) Brain single-cell suspensions of 9-month-old WT mice were generated for validation of microglial CD11c expression by flow cytometry. We first gate CD11b+ cells from single / live cells, followed by gating the CD11b+CD45low population as potential microglia. Although almost all cells in this population (~99%) are Tmem119+, CCR2 expression is undetectable, indicating that this CD11b+CD45low population is microglia. In contrast, CCR2 expression is detected in the CD11b+CD45high fraction, i.e., the putative macrophage population, whereas Tmem119 expression is undetectable. Analysis of CD11b+CD45low microglial populations using FMO negative controls on these FACS plots confirms the specificity. Brain CD45- cells, which mainly contain non-immune cells that do not express CD11c (neurons, astrocytes, oligodendrocytes, but not microglia), were used as negative controls to further validate the specificity of this FACS strategy. (B) The percentage of CD11c+ microglia in C57BL / 6 (B6) mice (n=3) during early development and normal aging was determined by flow cytometric analysis. CD11c+ microglia were first present during late embryonic life (E18.5), peaked during early postnatal life (P5), gradually declined to marginal levels during young adulthood (3 months of age), and then reappeared and expanded further during aging. (C) CD11c- microglia were isolated (>99% pure) from P5 B6-WT mice by negative selection with anti-CD11c magnetic beads and then co-incubated for 72 h at a 1:1 ratio in the presence or absence of pHrodo fluorescent dye-labeled apoptotic neurons (AN). After 72 h of incubation, 23% of all CD11c- microglia had taken up AN (AN+), of which 77.4% of AN+ microglia were CD11c+.In contrast, less than 1% of CD11c- microglia that did not receive AN (AN- reflects 77% of total microglia in culture at 72 h) express CD11c (upper panel). These findings indicate that approximately 18% of total CD11c- microglia express CD11c+ after incubation with AN, whereas 98% of microglia incubated for 72 h in the absence of AN remain CD11c- (lower panel). (D,E) CD11c- microglia isolated from P5 B6-WT mice were incubated for 24 h in the presence or absence of LPS (10 ng / mL) or Aβ (1 μM), followed by FACS analysis of microglial activation markers and CD11c expression. CD11c expression was assessed in CD86+ or MHC II+ activated microglia. Despite the upregulation of CD86 and MHC II in response to LPS and Aβ, no CD11c-microglial differentiation was observed in response to these stimuli. FACS plots are representative data from three experiments. (F) CD11c-microglia were isolated from P5 B6-WT mice by negative selection with anti-CD11c magnetic beads (>99% pure) and subsequently co-incubated for 72 h in the presence or absence of the αVβ3 integrin inhibitor Cilengitide (Cil) (10 μM) and / or the pan-TAM receptor inhibitor LDC1267 (1 μM) with or without pHrodo fluorescent dye-labeled AN at a 1:1 ratio. After 72 h of incubation, Cilengitide or LDC1267 reduced AN uptake by CD11c-microglia by approximately 50%, and the combination of these two inhibitors further inhibited AN uptake to background levels. Stimulation with AN induced CD11c expression in approximately 20% of early CD11c- microglia over a 72-h period, whereas CD11c expression was undetectable in the absence of AN stimulation during the same period. Inhibition of AN uptake by cilengitide or LDC1267 reduced the acquisition of a CD11c+ phenotype by CD11c- precursors by approximately 50-75% in each case, and inclusion of both inhibitors completely prevented this phenotypic transition (n=3).****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 by one-way ANOVA with Bonferroni's multiple comparison test. Data are presented as mean ± sem. [Diagram 2] Validation of microglial OPN expression by flow cytometry. Microglial OPN expression was validated by flow cytometry analysis in 9-month-old WT mice using a conventional intracellular staining protocol. Microglia were fixed and permeabilized with intracellular fixation and permeabilization buffer, followed by incubation with PE-conjugated anti-OPN Ab (1:10) for 30 min at 4 °C. Isotype control (1:10, PE-conjugated goat IgG) and OPN-KO microglia were used as negative controls. Microglia selectively expressing the intracellular isoform of OPN (OPN-i-KI) were used as positive controls. WT and OPN-i-KI microglia show similar levels of OPN staining, whereas staining of microglia from OPN-KO donors or microglia stained with isotype control does not yield a detectable signal. [Diagram 3]Figure 3A-H shows that the stability of CD11c expression by microglia is regulated by OPN. (A) Flow cytometry analysis of OPN expression in CD11c+ versus CD11c- microglia isolated from B6-WT mice at different stages of development and aging (n=3). OPN production was restricted to CD11c+ microglia during early development and normal aging. ****p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. (B) CD11c- microglia were isolated from P5 B6-WT mice and subsequently co-incubated with pHrodo fluorescent dye-labeled apoptotic neurons (AN) at a 1:1 ratio for 72 h. CD11c- microglia that phagocytosed AN differentiated into CD11c+ microglia with a sharp increase in the expression of OPN (n=3). ****p<0.0001 by two-tailed Student's t-test. (C, D) CD11c+ microglia (>95% pure) were isolated from P5 and 9-month-old WT and OPN-KO mice and subsequently incubated in conventional microglia culture medium (DMEM-F12 with 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / mL M-CSF) for 7 days. In vitro analysis of CD11c+ microglia stability was assessed by comparing the percentage of CD11c+ microglia on day 0 with day 7 (percentages were normalized to day 0). CD11c+ microglia in P5 WT and 9-month-old WT mice were stable, whereas OPN-KO CD11c+ microglia showed a significant loss of stability in P5 and 9-month-old WT mice (n=3). ****p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. (E, F) Ex vivo organotypic hippocampal slice cultures (OHSCs) were used to assess CD11c+ microglia stability. Freshly prepared OHSCs were incubated with 0.5 mg / mL clodronate liposomes at 35°C for 24 h to deplete endogenous microglia. Each microglia-free OHSC was supplemented with 4x103 CD11c+ microglia (>95% pure) isolated from P5 or 9-month-old WT or OPN-KO mice.Representative images showing CD11c+ microglia in hippocampal slices reconstituted with P5 WT or P5 OPN-KO CD11c+ microglia on day 7. CD11c+ microglia from P5 WT and 9-month-old WT mice stably expressed CD11c, whereas OPN deletion reduced CD11c expression by approximately 60% (n=3–4). Scale bar: 25 μm. ****p<0.0001, ***p<0.001 by two-way ANOVA with Bonferroni's multiple comparison test). (G,H) CD11c+ microglia isolated from 9-month-old WT and OPN-KO mice were incubated in vitro or in OHSCs for 7 days in the presence of 1 μM synthetic human Aβ1-42 peptide. CD11c+ microglia from 9-month-old WT mice were stable, whereas OPN deficiency led to a substantial reduction in the CD11c phenotype in the presence of Aβ in vitro and in OHSCs, respectively (n=3). ****p<0.0001, **p<0.01 by two-way ANOVA with Bonferroni's multiple comparison test. Data presented as mean ± sem. [Figure 4]Figure 4A-B shows that OPN deletion reduces the percentage of CD11c+ microglia. (A) The percentage of CD11c+ and CD11c- microglia in WT and OPN-KO mice during early development and aging was determined by flow cytometry. OPN deletion led to a significant decrease in the percentage of CD11c+ microglia in WT mice at P5, 6 months of age, and 9 months of age. CD11c isotype control was used as a negative control (n=3). **p<0.01, ****p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. (B) The percentage of CD11c+ and CD11c- microglia was analyzed in brain cryosections of WT and OPN-KO mice at P5 and 9 months of age. OPN-KO mice showed a significantly decreased percentage of CD11c+ microglia compared to WT mice (n=3). The proportion of CD11c+ or CD11c- microglia was assessed by the percentage of CD11c+ (CD11c+Iba-1+) or CD11c- microglia (CD11c-Iba-1+) among total microglia (Iba-1+) in each field. Brain slides incubated without anti-CD11c primary Ab or tyramide signal amplification (TSA) were included as negative controls to exclude nonspecific immunofluorescence signals. *p<0.05 by two-tailed Student's t-test. Data are shown as mean ± sem. [Diagram 5] Figure 5A-B shows that the number of CD11c+ microglia is comparable in WT and OPN-KO mice at day 0 in vitro and in OHSCs. (A, B) The number of CD11c+ microglia in P5, 9-month-old WT mice was compared to age-matched OPN-KO mice by flow cytometry (in vitro) and immunofluorescence staining (in OHSCs) at day 0. The number of CD11c+ microglia was similar between P5, 9-month-old WT and OPN-KO mice at day 0 in vitro and in OHSCs (n=3). Statistical analysis was performed by two-tailed Student's t-test. [Figure 6]Validation of CD11c expression by microglia subjected to cold isolation by flow cytometry. To validate microglial CD11c expression, brain single-cell suspensions from 9-month-old WT mice were generated by cold isolation. CD11b+ cells were gated on single / live cells, followed by gating CD11b+CD45low cells as microglia and CD11b+CD45hi as macrophages. The majority of cells in the CD11b+CD45low population (95%) are Tmem119+, but CCR2 expression is undetectable, indicating that the CD11b+CD45low population is microglia. In contrast, CCR2 expression is detected in CD11b+CD45high macrophages, but Tmem119 expression is not. Analysis of this CD11b+CD45low microglial population using FMO negative controls confirmed the specificity of CD11c staining. Brain CD45 − cells, which primarily contain non-immune cells that do not express CD11c, were included as a negative control. [Figure 7]Figure 7A-G shows the definition of the intrinsic gene program of CD11c+ microglia. (A) Transcriptome profiling of CD11c+ and CD11c- microglia was analyzed in P5 and 9-month-old WT mice by RNA-seq. Venn diagrams show the number of genes expressed in P5 and 9-month-old WT CD11c+ and CD11c- microglia, as well as genes exclusively expressed by each microglial subset. Unique genes in CD11c+ microglia were identified within the top 0.15% of genes showing fold change and raw counts, negatively expressed by their CD11c- counterparts. The threshold of negative expression was defined according to the raw counts of genes with no expression, e.g., Itgax in CD11c- microglia. A similar method was used to identify unique genes in CD11c- microglia. (B) The CD11c+ microglial core gene signature was identified as overlapping unique genes in P5 and 9-month-old CD11c+ microglia. (C) Heatmap showing four CD11c+ microglial core genes, including Itgax, Cd209a, Cd209f, and Cd36, in P5 and 9-month-old CD11c+ microglia compared to their CD11c- counterparts (FDR<0.05). (D,E) Validation of core genes at protein level. CD11c+ microglial core genes were validated at the protein level by flow cytometry analysis. Expression of surface proteins CD36 and CD209a was exclusively expressed by CD11c+ microglia from P5 WT and 9-month-old WT mice compared to CD11c- microglia (n=3). ****p<0.0001, **p<0.01 by two-tailed Student's t-test. (F) CD11c- microglia isolated from P5 WT mice were incubated with LPS (10 ng / mL) or Aβ (1 μM) for 24 h, followed by flow cytometry analysis of CD36 and CD209 expression. Protein expression of these core genes was barely detectable in CD86+ activated CD11c- microglia in response to activation stimuli. FACS plots are representative of three experiments. (G) In vitro differentiation of CD11c+ microglia.CD11c+ and CD11c- microglia were freshly isolated from P5 WT mice. CD11c+ microglial phenotype was verified by analyzing mRNA expression of representative core genes on day 0. CD11c- microglia were then incubated with apoptotic neurons (AN) for 72 hours to induce differentiation of CD11c- microglia into CD11c+ microglia. Core genes were verified in CD11c+ microglia (differentiated) compared to CD11c- microglia (undifferentiated) on day 3 by Q-PCR. CD11c+ microglial phenotype expressed in CD11c+ microglia on day 0 is indicated by upregulation of selected core genes compared to CD11c- microglia. AN-induced CD11c- microglial differentiation is accompanied by induction of the CD11c+ microglial gene program. Core gene expression was normalized to z-score (p<0.05) as shown in the heatmap (n=3). [Figure 8]Figure 8A-G shows that OPN regulates the intrinsic function of CD11c+ microglia. (A) Microglia isolated from P5 and 9-month-old WT, OPN-KO and OPN-i-KI mice were incubated with 136 mg of pHrodo Green-labeled synaptosomes per 1 × 105 cells for 1 h, followed by flow cytometry analysis of CD11c+ microglial phagocytosis of synaptosomes. CD11c+ microglia from P5, but not 9-month-old WT mice, were highly phagocytic to phagocytose synaptosomes in vitro. Deletion of OPN-s significantly suppressed this function, whereas OPN-i was not involved in this process (n=3). ****p<0.0001 (by one-way ANOVA with Bonferroni's multiple comparison test). (B) Ex vivo analysis shows that CD11c+ microglia from P5 WT mice, but not 9-month-old WT mice, show robust activity in phagocytosis of synaptic proteins. P5 WT mice lacking OPN-s, but not OPN-i, showed a substantial reduction in the uptake of presynaptic protein synaptophysin or postsynaptic protein PSD95 by CD11c+ microglia (n=3). ****p<0.0001, ***p<0.001 by two-tailed Student's t-test. (C) CD11c+ microglia expressed high levels of the proliferation marker Ki-67 during early development (P5). OPN-s deletion, but not OPN-i deletion, reduced Ki-67 expression in CD11c+ microglia (n=3). *p<0.05 by one-way ANOVA with Bonferroni's multiple comparison test. (D) During aging, CD11c+ microglia exhibited relatively low levels of proliferative activity, and CD11c+ microglial Ki-67 expression was substantially reduced in mice lacking OPN-s (n=3). *WT vs. OPN-KO, # WT vs. OPN-i-KI. ****p<0.0001, *p<0.05, ####p<0.0001, #p<0.05 by two-way ANOVA with Bonferroni's multiple comparison test.(E) Microglia isolated from 9-month-old WT mice were incubated for 24 h in the presence or absence of 12.5 μg / mL recombinant mouse OPN (rmOPN) or 10 μM αVβ3 inhibitor (Cilengitide), followed by flow cytometric analysis of the proliferation marker Ki-67. rmOPN enhanced Ki-67 expression in CD11c+αVβ3+ microglia from 9-month-old WT mice, whereas this effect was completely suppressed by the αVβ3 inhibitor (n=3). **p<0.01, *p<0.05 (by one-way ANOVA with Bonferroni's multiple comparison test). (F) Expression of TNF-α was barely detectable in CD11c+ microglia from P5 mice, whereas its expression gradually increased during aging. Age-dependent increase in TNF-α expression in CD11c+ microglia was significantly reduced in WT mice lacking OPN-s, but not OPN-i (n=3). *WT vs OPN-KO, # WT vs OPN-i-KI. ****p<0.0001, ####p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. (G) Microglia isolated from 9-month-old WT mice were incubated with or without 12.5 μg / mL recombinant mouse OPN (rmOPN) or 10 μM αVβ3 inhibitor (Cilengitide) for 24 h, followed by flow cytometric analysis of TNF-α expression. The upregulation of TNF-α expression induced by rmOPN was completely abolished by the αVβ3 inhibitor in CD11c+αVβ3+ microglia of 9-month-old WT mice (n=3). **p<0.01 by one-way ANOVA with Bonferroni's multiple comparison test. Data are presented as mean±sem. [Figure 9] Figure 1 shows that microglial αVβ3 expression gradually increased in WT mice during aging. Representative FACS plots and summary of microglial αVβ3 expression in P5, P30, 3-month-old, 6-month-old, and 9-month-old WT mice. Microglial αVβ3 expression gradually increased during aging. Representative plots are from four independent experiments. [Figure 10]Figure 10A-D shows that the CD11c+ microglial core gene program and OPN-dependent stable phenotype are retained in the 5XFAD disease setting. (A, B) Selected core genes of CD11c+ microglia were validated at the protein level by flow cytometry. Protein expression of CD209a and CD36 was exclusively expressed by CD11c+ microglia of 9-month-old 5XFAD mice compared to CD11c- microglia. Isotype controls for CD36 and CD209a were used as negative controls. (n=3). ****p<0.0001 by two-tailed Student's t-test. (C) CD11c+ microglia isolated from 9-month-old 5XFAD or OPN-KO.5XFAD mice were cultured in vitro or in OHSCs for 7 days. CD11c+ microglia from 9-month-old 5XFAD mice were stable both in vitro and in OHSCs, whereas the stability of CD11+ microglia from OPN-KO.5XFAD mice was rapidly reduced by approximately 40%-80% in vitro and in OHSCs, respectively (n=3). ****p<0.0001, ***p<0.001 by two-way ANOVA with Bonferroni's multiple comparison test. (D) CD11c+ microglia isolated from 9-month-old 5XFAD and OPN-KO.5XFAD mice were cultured in vitro or in OHSCs for 7 days in the presence of 1 μM synthetic human Aβ1-42 peptide. CD11c+ microglia from 9-month-old 5XFAD mice were stable, but OPN deficiency led to a sharp decrease in CD11c+ microglial stability in response to Aβ stimulation in vitro and in OHSCs (n=3–4). ***p<0.001, **p<0.01 by two-way ANOVA with Bonferroni's multiple comparison test. Data are presented as mean ± sem. [Figure 11]Figure 11A-L shows that OPN deletion reduced AD pathology and rescued cognitive deficits in 5XFAD mice. (a) Flow cytometry analysis of OPN expression in microglia (CD11b+), astrocytes (GFAP+) and neurons (MAP2+) of 5XFAD mice at different stages of disease development. Microglia are the main cellular source of OPN production in the brain of 5XFAD mice during disease progression (n=3). (b, c) Microglial OPN expression at the mRNA level (RT-qPCR) and protein level (flow cytometry). Microglial OPN expression was increased at both the mRNA and protein levels in 5XFAD mice compared to age-matched WT mice during disease progression (n=3-4). ****p<0.0001, ***p<0.001, *p<0.05 by two-way ANOVA with Bonferroni's multiple comparison test. (d) Flow cytometry analysis of OPN expression in CD11c+ and CD11c- microglia from 5XFAD mice at different disease stages. OPN production is restricted to CD11c+ microglia during 5XFAD disease progression (n=3). ****p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. (e) Flow cytometry analysis showing the percentage of CD11c+OPN+ microglia was increased in 5XFAD mice compared to age-matched WT mice during disease progression (n=3). ****p<0.0001, **p<0.01 by two-way ANOVA with Bonferroni's multiple comparison test. (f) Ex vivo analysis of microglial TNF-α expression in WT, 5XFAD and OPN-KO.5XFAD mice during disease progression. OPN deletion reduced the disease-dependent increase in microglial TNF-α production in 5XFAD mice (n=3). *WT vs. 5XFAD, ****p<0.0001; #5XFAD vs. OPN-KO.5XFAD, ####p<0.0001 by two-way ANOVA with Bonferroni's multiple comparison test. (g,h) Representative immunofluorescence images and quantification of Aβ area in brain cryosections of 5XFAD and OPN-KO.5XFAD mice at different stages of disease.OPN deletion resulted in a substantial reduction in total Aβ plaque area in the cortex and hippocampus of 5XFAD mice during disease progression (n=3-4). ****p<0.0001, *p<0.05, ns: not significant (by two-way ANOVA with Bonferroni's multiple comparison test). Scale bar=200 μm. (i) Different morphologies of Aβ plaques were stained with anti-Aβ (6E10) and Thio-S in brain cryosections of 9-month-old 5XFAD and OPN-KO.5XFAD mice. OPN deletion resulted in a reduction in the percentage of diffuse plaques (6E10+Thio-S-) within total plaques (6E10+) in 5XFAD mice (n=3). ***p<0.001 by two-tailed Student's t-test. (j, k) Representative immunofluorescence images and quantification of dystrophic neurites (labeled with N-terminal APP) per plaque (labeled with Thioflavin-S) in brain sections from 5XFAD and OPN-KO.5XFAD mice. The number of dystrophic neurites was quantified within a 25 μm area of ​​each plaque. The number of dystrophic neurites per plaque was significantly reduced in OPN-KO.5XFAD mice compared to 5XFAD mice (n=30-40 plaques from 3 mice per group). ****p<0.0001, ***p<0.001, ns: not significant (by two-way ANOVA with Bonferroni's multiple comparison test). Scale bar=200 μm. (l) Spatial learning and memory (acquisition trials) and cognitive flexibility (reversal trials) were assessed by water T-maze in 9-month-old 5XFAD and OPN-KO.5XFAD mice. Age-matched WT mice were induced as controls. 5XFAD mice lacking OPN showed an increased percentage of correct choices in both acquisition and reinstatement trials (n=3-5, #:WT vs. 5XFAD, #p<0.05, ##p<0.01, ###p<0.001. *5XFAD vs. OPN-KO.5XFAD, **p<0.01). Statistical analysis was performed by two-way ANOVA with Bonferroni's multiple comparison test. All data are presented as mean ± sem. [Figure 12]Figure 12A-B shows confirmation of OPN KO in OPN-KO.5XFAD mice. (a) PCR genotyping results verify successful knockout of OPN in OPN-KO.5XFAD mice. 5XFAD mice (OPNWT) show OPNWT band at 300bp. After crossing with OPN-KO mice, the resulting OPN-KO.5XFAD mice obtained OPN KO band at 500bp. All mice show 5XFAD transgene band at 377bp and internal positive control IL-2 band at 324bp. (b) Validation of OPN KO at protein level in microglia of 9-month-old OPN-KO.5XFAD mice. Microglial expression of OPN was undetectable in OPN-KO.5XFAD mice. Microglia from age-matched 5XFAD mice were used as positive control cells while isotype control was used as negative control. Contour plots were representative results of three independent experiments. [Figure 13] Validation of microglial CD11c expression in 5XFAD mice by flow cytometry. Flow cytometry validation of microglial CD11c expression in 9-month-old 5XFAD mice. CD11b+ cells were gated on single / live cells, followed by gating on CD11b+CD45low as microglia and CD11b+CD45hi as macrophages. The majority (~90%) of the CD11b+CD45low population is Tmem119+, but CCR2 expression is undetectable, confirming the CD11b+CD45low population as microglia. In contrast, CCR2 expression is detected in CD11b+CD45high macrophages, but Tmem119 expression is not. Analysis of this CD11b+CD45low microglial population with the FMO negative control confirms the specificity of the CD11c staining. Brain CD45 − cells, which primarily contain non-immune cells that do not express CD11c, were included as a negative control. [Figure 14]Figure 14A-L shows that OPN production by pathogenic CD11c+ microglia promotes pro-inflammatory responses and inhibits Aβ uptake in 5XFAD mouse brain. (a) Heatmap showing representative differentially expressed genes (DEGs) in CD11c+ microglia from 9-month-old 5XFAD mice compared to OPN-KO.5XFAD mice. OPN deletion in CD11c+ microglia resulted in downregulation of inflammatory response-related genes (e.g., Tnfrsf9, Il1b, Ifitm1, Ccl17) and upregulation of phagocytosis-related genes (e.g., Trem2, Axl, Mertk, Cd68). Upregulation: log2FC>0.5, downregulation: log2FC<-0.5, FDR<0.05. (b) The percentages of CD11c+OPN+, CD11c+OPN-, CD11c- OPN+ and CD11c- OPN- microglial subsets in the whole brain of 9-month-old 5XFAD mice were determined by flow cytometry and calculated as the number of each subset within the number of total microglia in the brain (n=3). ****p<0.0001 by one-way ANOVA with Bonferroni's multiple comparison test. (c) The proportions of CD11c+OPN+, CD11c+OPN- and CD11c- OPN- microglial subsets were analyzed by immunofluorescence staining in brain cryosections of 9-month-old 5XFAD mice. Cortex / hippocampus (C / H): The percentage of each microglial subset was calculated as its number in the C / H region out of its total number in the whole brain (90 fields from 3 mice were analyzed). Within the C / H region, the percentage of each microglial subset in the periplaque region (within 25 μm of the Aβ plaque core) was calculated as the number of each microglial subset located in the periplaque region out of its total number in the whole brain (33 fields from 3 mice were analyzed). The percentage of plaque-distal microglia was calculated as the number of each microglial subset located >25 μm of the Aβ plaque core out of its total number in the whole brain (33 fields from 3 mice were analyzed). Within the periplaque region, the Aβ-uptake cells of each microglial subset were calculated as the number of Aβ+-uptake microglia out of the total number of each microglial subset present in the whole brain (33 ROIs from 3 mice were analyzed).***p<0.001, ns: not significant (by one-way ANOVA with Bonferroni's multiple comparison test). (d) Pie charts summarize the percentages of CD11c+OPN+, CD11c+OPN- and CD11c- OPN- microglial subsets in the brains of 9 mo 5XFAD mice. Pie charts were representative results of three independent experiments. (e, f) Representative flow cytometry plots and quantification of TNF-α and TREM2 expression in CD11c+OPN+ and CD11c+OPN- microglia from 9 month old 5XFAD mice. CD11c+OPN+ microglia produced significant amounts of TNF-α but showed reduced levels of TREM2 expression compared to CD11c+OPN- microglia (n=3). ****p<0.0001, **p<0.01 (two-tailed Student's t-test). (g) Flow cytometric analysis of TREM2 expression in CD11c+TNF-α+ vs. CD11c+TNF-α- microglia from 9-month-old 5XFAD mice. TREM2 expression was significantly lower in CD11c+TNF-α- microglia than in CD11c+TNF-α- microglia (n=3). ***p<0.001, (two-tailed Student's t-test). (h) Flow cytometric analysis of αVβ3 expression in CD11c+OPN+, CD11c+OPN- and CD11c-OPN- microglial subsets from 9-month-old 5XFAD mice. CD11c-OPN- microglia do not express substantial amounts of αVβ3, whereas CD11c+OPN+ and CD11c+OPN- microglial subsets express similar levels of αVβ3 (n=3). ****p<0.0001, ns: not significant (by one-way ANOVA with Bonferroni's multiple comparison test). (i) Quantification of Aβ-ingesting CD11c+ microglia in brain cryosections from 9-month-old 5XFAD and OPN-KO.5XFAD mice. Aβ uptake was calculated as the number of Aβ+-ingesting CD11c+ microglia within the total number of CD11c+ microglia surrounding plaques (24 ROIs from 3 mice were analyzed per group). ****p<0.0001 (by two-tailed Student's t-test).(j) Flow cytometry analysis of CD11c+ microglial TNF-α production in 9-month-old 5XFAD and OPN-KO.5XFAD mice. CD11c+ microglial TNF-α expression was substantially reduced in OPN-KO.5XFAD mice compared to age-matched 5XFAD (n=3). ***p<0.001 by two-tailed Student's t-test. (k) Flow cytometry analysis showing production of TNF-α in CD11c+αVβ3+ versus CD11c+αVβ3- microglia in 9-month-old 5XFAD and OPN-KO.5XFAD mice. OPN deletion resulted in a significant reduction in TNF-α in CD11c+αVβ3+ but not CD11c+αVβ3- microglia in 5XFAD mice (n=3). ****p<0.0001, ns: not significant (by one-way ANOVA with Bonferroni's multiple comparison test). (l) Flow cytometry analysis of TREM2 expression by CD11c+ microglia in 9-month-old 5XFAD and OPN-KO.5XFAD mice. OPN deficiency resulted in a significant increase in TREM2 expression in CD11c+ microglia in 5XFAD mice (n=3). **p<0.01 (by two-tailed Student's t-test). [Figure 15] FIG. 15A-B shows immunofluorescence staining of CD11c microglial subsets in brain cryosections of 5XFAD mice. (a) Immunofluorescence signals of microglial CD11c expression were verified in 9-month-old 5XFAD mice. Brain cryosections incubated without anti-CD11c primary Ab or tyramide signal amplification (TSA) reagent were used as negative controls. Scale bar = 50 μm. (b) Representative immunofluorescence staining of CD11c+OPN+ microglia (CD11c+OPN+Iba-1+, indicated by cyan arrows), CD11c+OPN- microglia (CD11c+Iba-1+, indicated by magenta arrows) and CD11c-OPN- microglia (Iba-1+, indicated by yellow arrows) in the brains of 9-month-old 5XFAD mice. Scale bar = 25 μm. [Figure 16]Figure 16A-E shows microglial expression of TREM2, TNF-α and canonical OPN receptor. (a) Representative flow cytometry plots and quantification of TREM2 mean fluorescence intensity (MFI) in CD11c+OPN+ and CD11c+OPN- microglial subsets from 9-month-old 5XFAD mice. The CD11c+OPN- microglial subset showed an increased percentage of TREM2+ cells compared to the CD11c+OPN+ subset, while TREM2 MFI was comparable between these two microglial subsets (n=3). Statistical analysis was performed by two-tailed Student's t-test. (b) Representative flow cytometry plots of TNF-α and TREM2 expression in CD11c-OPN- microglial subsets from 9-month-old 5XFAD mice. CD11c-OPN- microglia expressed low levels of TNF-α and negligible levels of TREM2. Plots were representative results of three independent experiments. (c) Microglial expression of canonical OPN receptors in 5XFAD mice at different stages of disease. Expression of αVβ3 gradually increased (n=3), whereas expression of CD44 or αVβ5 was barely detectable in microglia of 5XFAD mice during disease progression. Histograms of CD44 and contour plots of αVβ3 and αVβ5 were representative results of three independent experiments. (d) Effect of OPN-αVβ3 interaction on CD11c+ microglial production of TNF-α. Upregulated TNF-α production induced by rmOPN was completely suppressed by αVβ3 inhibitor in CD11c+ microglia of 9-month-old OPN-KO.5XFAD mice (n=3). ****p<0.0001 by one-way ANOVA with Bonferroni's multiple comparison test. (e) Representative flow cytometry plots and quantification of TREM2 MFI in CD11c+ microglia from 9-month-old 5XFAD and OPN-KO.5XFAD mice (n=3). OPN-KO.5XFAD mice showed an increased percentage of TREM2+ microglia within CD11c+ microglia compared to 5XFAD mice, but TREM2 MFI was comparable between the two groups. Two-tailed Student's t-test was performed for statistical analysis. [Figure 17] The proportion of microglial subsets in the 5XFAD mouse brain is shown. Schematic diagram showing that less than 30% of CD11c-OPN- microglia are enriched in the cortical / hippocampal (C / H) region of the 5XFAD mouse brain, and only 13% are located in the periplaque region. They have a low capacity for Aβ uptake (about 2%) and express marginal levels of TNF-α or TREM2. The majority of CD11c+OPN- microglia (about 70%) are present in the C / H region. They are highly enriched in the periplaque region (63%) and may be protective, since most of them (about 60%) in the periplaque region can uptake Aβ but can only produce negligible levels of TNF-α, while expressing high levels of TREM2. In contrast, the CD11c+OPN+ microglial subset, which is also significantly enriched in periplaque regions (~60%), exhibits low levels of Aβ uptake (~7%), but nearly 60% produce TNF-α and express low levels of TREM2, supporting the notion that CD11c+OPN+ microglia may be a pathogenic microglial subset. [Figure 18]Figure 18A-D shows in vitro analysis of OPN-dependent inhibition of the TREM2-lysosomal phagocytosis pathway. (a) Protocol for in vitro analysis of OPN-dependent inhibition of the TREM2-lysosomal phagocytosis pathway in 9-month-old 5XFAD and OPN-KO.5XFAD mice. (b) TREM2 expression was significantly higher in CD11c+ microglia of OPN-KO.5XFAD mice compared to 5XFAD mice, and its expression was rapidly decreased in response to rmOPN. Neutralizing anti-OPN Ab inhibited the effect of rmOPN (n=3). ****p<0.0001, ***p<0.001, **p<0.01 (by one-way ANOVA with Bonferroni's multiple comparison test). (c) CD68 expression in CD11c+ microglia of OPN-KO.5XFAD mice was higher than that of 5XFAD mice, and the expression was significantly decreased by rmOPN, whereas anti-OPN Ab abolished the effect of rmOPN (n=3). ****p<0.0001, ***p<0.001 (by one-way ANOVA with Bonferroni's multiple comparison test). (d) The MFI of FAM-Aβ1-42 after 1 hour of incubation was determined by flow cytometry in the lysosomes of CD11c+ microglia (CD11c+CD68+) and defined as AβMFI1h. The MFI of FAM-Aβ retained in the lysosomes of CD11c+ microglia (CD11c+CD68+) 24 hours after FAM-Aβ1-42 removal was determined and defined as AβMFI24h. CD11c+ microglia from OPN-KO.5XFAD mice showed a higher Aβ degradation rate [(AβMFI1h-AβMFI24h) / AβMFI1h] than 5XFAD mice, but rmOPN significantly reduced the Aβ degradation rate. The effect of rmOPN was completely blocked by anti-OPN Ab (n=3). **p<0.01, *p<0.05 by one-way ANOVA with Bonferroni's multiple comparison test. All data are shown as mean ± sem. [Figure 19]Figure 19A-J shows that OPN impairs Aβ plaque compaction via suppression of the TREM2-lysosomal phagocytosis pathway. (a, b) Representative immunofluorescence images and quantification of CD11c+TREM2+ microglia in brain cryosections of 9-month-old 5XFAD and OPN-KO.5XFAD mice. The percentage of CD11c+TREM2+ microglia was 2-fold higher in OPN-KO.5XFAD mice compared to 5XFAD mice (n=14 ROIs from 3 mice / group). *p<0.01 by two-tailed Student's t-test. Scale bar=50 μm. (c, d) Representative immunofluorescence images and quantification of CD68+ area of ​​CD11c+ microglia located within 25 μm of Aβ plaques (defined as plaque-associated CD11c+ microglia) in 9-month-old 5XFAD and OPN-KO.5XFAD mice. OPN deletion resulted in an increase in CD68+ area in CD11c+ microglia of 5XFAD mice (n=20 ROIs from 3 mice / group). *p<0.05 by two-tailed Student's t-test. Scale bar=25 μm. (e, f) Representative immunofluorescence images and quantification of Cathepsin B+ area in plaque-associated CD11c+ microglia of 9-month-old 5XFAD and OPN-KO.5XFAD mice. A substantial increase in Cathepsin B+ area was observed in CD11c+ microglia of OPN-KO.5XFAD compared to 5XFAD (n=22 ROIs from 3 mice / group). ****p<0.0001 by two-tailed Student's t-test. Scale bar=25 μm. (g) Representative immunofluorescence images showing two different forms of Aβ plaques in 9-month-old 5XFAD and OPN-KO.5XFAD mouse brains stained with 6E10 (red) and Thioflavin-S (green). Total plaques were defined as 6E10+. White arrows indicate diffuse plaques (6E10+Thio-S-) and compact plaques (6E10+Thio-S+) are indicated by yellow arrows. Scale bar = 100 μm. (h, i) Quantification of the total area (6E10+) and compact area (6E10+Thio-S+) of each plaque in 3-, 6-, and 9-month-old 5XFAD and OPN-KO.5XFAD mice (n = 30–40 compact plaques from 3 mice / group).(by two-way ANOVA with Bonferroni's multiple comparison test) ****p<0.0001, *p<0.05, ns: not significant. (j) Compaction index of each compact plaque in 3-, 6-, and 9-month-old 5XFAD and OPN-KO.5XFAD mice. Compaction index was calculated as 6E10+Thio-S+area / 6E10+area (n=30-40 compact plaques from 3 mice / group). ****p<0.0001, **p<0.01, ns: not significant (by two-way ANOVA with Bonferroni's multiple comparison test). All data are shown as mean ± sem. [Figure 20]Figure 20A-I shows that increased microglial OPN production positively correlates with AD severity in human AD brains. (a) OPN protein expression in brain (middle frontal gyrus) homogenates of AD patients (CDR ≥ 1, n = 11), mild cognitive impairment patients (MCI, CDR 0.5, n = 10) and cognitively normal controls (CDR 0, n = 11). AD patients showed significantly higher brain OPN protein expression compared to cognitively normal controls, whereas OPN expression was comparable between MCI and AD patients and between MCI and controls. **p < 0.01, ns: not significant (by one-way ANOVA with Bonferroni's multiple comparison test). (b) Positive correlation between brain OPN expression and CDR score in AD patients (CDR ≥ 1, n = 11), MCI patients (CDR 0.5, n = 10) and controls (CDR 0, n = 11). r=0.5046, p=0.0032 (by Pearson's correlation analysis). (c, d) Representative immunofluorescence images of the middle frontal gyrus of AD patients and normal controls stained for Iba-1 (microglia, red), CD11c (green) and OPN (cyan) indicated by white arrows. The percentage of CD11c+OPN+ microglia was significantly higher in brains of AD patients (CDR ≥ 1, n = 8) compared to MCI (CDR 0.5, n = 9) and control subjects (CDR 0, n = 5). Each dot represents the mean percentage of CD11c+OPN+ microglia relative to total Iba-1+ cells in 10-12 fields of view of each brain sample. Scale bar = 25 μm. ****p<0.0001, ns: not significant (by one-way ANOVA with Bonferroni's multiple comparison test). (e) The percentage of CD11c+OPN+ microglia in the brain positively correlates with CDR scores in AD patients (CDR ≥ 1, n = 8), MCI patients (CDR 0.5, n = 9) and controls (CDR 0, n = 5). r = 0.8383, p < 0.0001 by Pearson's correlation analysis. (f, g) Brain OPN concentration and the percentage of CD11c+OPN+ microglia positively correlate with neuritic plaque assessment by Pearson's correlation analysis (r = 0.4919, p = 0.0043; r = 0.8226, p < 0.0001, respectively, by Pearson's correlation analysis).(h, i) Brain OPN levels and the percentage of CD11c+OPN+ microglia positively correlate with tangle scores by Pearson's correlation analysis (r=0.4884, p=0.0046; r=0.7434, p<0.0001, respectively, by Pearson's correlation analysis). All data are shown as mean ± sem. [Figure 21] FIG. 1 shows brain penetration of unmodified vs. modified KK-Ahx-Angiopep2-conjugate under one embodiment. [Figure 22] 1 shows an exemplary Angiep-2 conjugated anti-OPN / anti-CD11b mAb under one embodiment. [Diagram 23] An exemplary model showing how OPN may function in Alzheimer's disease: OPN inhibition of microglial Aβ plaque compaction and promotion of inflammatory responses contributes to cognitive impairment. [Figure 24] Figures 24A-C show exemplary data correlating OPN expression with disease progression in the 5XFAD mouse model. As shown, (A) microglia are the major cell source producing OPN in the brain of 5XFAD mice during disease progression (n=3). In (B) and (C), microglial OPN expression was increased at both the mRNA and protein levels in mice compared to age-matched wild-type mice during disease progression (n=3-4). ****p<0.0001, ***p<0.001, *p<0.05 by two-way ANOVA with Bonferroni's multiple comparison test. These figures are the same as Figures 11A, 11B, and 11C, respectively. [Diagram 25]Figures 25A-B show exemplary data of OPN expression in CD11c+ microglia in the 5XFAD mouse model. As shown, (A) OPN production is restricted to CD11c+ during 5XFAD disease progression (n=3). ****p<0.0001. (B) The percentage of CD11c+OPN+ microglia increased in 5XFAD mice compared to age-matched wild-type mice during disease progression (n=3). ****p<0.0001, **p<0.01 by two-way ANOVA with Bonferroni's multiple comparison test. These figures are the same as Figures 11D and 11E, respectively. [Figure 26] Figure 26A-F shows exemplary data of OPN deletion reducing inflammatory response, plaque area and diffuse, neuritic dystrophy and cognitive impairment in 5XFAD mouse model. As shown, (A) deletion of OPN reduced microglial TNF-α production (n=3, *WT vs. 5XFAD, #5XFAD vs. OPN- / -.5XFAD, ****p<0.0001, #####p<0.0001). (B)(C) OPN deletion resulted in a reduction in Aβ plaque area in total plaques (6E10+) and (D) the percentage of diffuse plaques (6E10+Thio-S-) in 5XFAD mice (n=3). (E) OPN deletion reduced the number of dystrophic neurites in 5XFAD mice (n=3). (F) Genetic depletion of OPN rescued cognitive impairment in 5XFAD mice (n=3-5, #:WT vs. 5XFAD, #p<0.05, ##p<0.01, ####p<0.001, ####p<0.0001, *5XFAD vs. OPN- / -.5XFAD, **p<0.01). Analyzed by two-way ANOVA with Bonferroni's multiple comparison test. These figures are the same as Figure 11F, Figure 11G, Figure 11H, Figure 11I, Figure 11K, and Figure 11I, respectively. [Figure 27]Figure 27A-E shows exemplary data of OPN promoting inhibition of pro-inflammatory and phagocytic responses by CD11c+ microglia in 5XFAD mice. (A) OPN deletion in CD11c+ microglia resulted in downregulation of inflammatory response-related genes and upregulation of phagocytosis-related genes in 9-month-old 5XFAD mice as shown by heatmaps. (log2FC>1 or log2FC<-1, FDR<0.05). (B) The number of TNF-α expressing CD11c+ microglia was substantially reduced in OPN- / -.5XFAD mice compared to advanced disease 5XFAD (n=3). ****p<0.0001, ***p<0.001. (C) OPN deletion resulted in a significant reduction in TNF-α expression in CD11c+αVβ3+ microglia, but not in CD11c+αVβ3- microglia, of 5XFAD mice (n=3). (By two-way ANOVA with Bonferroni's multiple comparison test) ****p<0.0001, *p<0.05. (D)(E) In situ analysis of brain cryosections revealed that Aβ phagocytosed by CD11c+ microglia was reduced in 9-month-old 5XFAD compared to OPN- / -.5XFAD mice. Yellow arrows indicate Aβ material internalized inside microglia. The ratio of Aβ phagocytosis was calculated as the Aβ+ area phagocytosed by CD11c+ microglia (20 CD11c+ microglia from 3 mice). (By two-tailed Student's t-test) ***p<0.001. FIG. 27A is the same as FIG. 14A. [Figure 28]Figure 28A-E shows exemplary data of OPN suppressing the TREM2-Axl-lysosomal pathway in CD11c+ microglia from 5XFAD mice. (A) TREM2 expression in CD11c+ vs. CD11c- microglia was analyzed by FACS in 5XFAD mice during disease progression as indicated. TREM2 was exclusively expressed by CD11c+ microglia (n=3). ****p<0.0001, *p<0.05. (B)(C) The percentage of CO11c+TREM2+ microglia was 2-fold higher in OPN- / -.5XFAD mice compared to 5XFAD mice (n=6 sections from 3 mice / group). **p<0.01. (D) OPN deficiency resulted in an increase in CD68+ area in plaque-associated CD11c+ microglia in both the cortex and hippocampus of 9-month-old 5XFAD mice (cortex: 30 plaques from 6 sections from 3 mice, hippocampus: 20 plaques from 6 sections from 3 mice). *p<0.05. (E) A substantial increase in cathepsin B+ area was observed in CD11c+ microglia in both the cortex and hippocampus of OPN- / -.5XFAD compared to 5XFAD (cortex: 22 plaques from 3 mice, hippocampus: 11 plaques from 3 mice). ****p<0.0001, '**p<0.01 by two-tailed Student's t-test. Figure 28B is the same as Figure 19A. Figure 28C is the same as Figure 19B. Figure 28D is the same as Figure 19D. Figure 28E is the same as Figure 19E. [Figure 29]Figure 29A-D shows exemplary data of OPN inhibiting CD11c+ microglial compaction of Aβ plaques in 5XFAD mice. (A) Brains of 9 month old 5XFAD and OPN- / -.5XFAD mice stained with 6E10 (red) and thioflavin-S (green) showing two different morphologies of Aβ plaques. White arrows indicate diffuse plaques (6E10+Thio-S-) and yellow arrows indicate dense plaques (6E10+Thio-S-). (B)(C) Quantification of total area (6E10+) and dense area (6E10+Thio-S+) of each plaque in 3 month, 6 month and 9 month old 5XFAD and OPN- / -.5XFAD mice (30-40 dense plaques from 3 mice per group). (D) Compaction index of each compact plaque in 3-, 6-, and 9-month-old 5XFAD and OPN- / -.5XFAD mice. Compaction index was calculated as 6E10+Thio-S+ area / 6E10+ area (30-40 compact plaques from 3 mice per group). (by two-way ANOVA with Bonferroni's multiple comparison test) ****p<0.0001, **p<0.01. Figures 29A-D are the same as Figures 19G-J, respectively. [Diagram 30] 30A-B show exemplary data correlating brain OPN levels with Alzheimer's disease severity in human brain. (A) OPN protein expression in middle frontal gyrus homogenates of AD patients (CDR≧1, n=11), mild cognitive impairment patients (MCI, CDR 0.5, n=10) and cognitively normal controls (CDR 0, n=11). Brain OPN protein expression was increased in AD patients compared to normal controls. **p<0.01 by one-way ANOVA with Tukey's multiple comparison test. (B) Positive correlation between brain OPN expression levels and CDR scores in AD patients. MCI patients and controls. r=0.5046, p=0.0032 by Pearson's correlation analysis. FIG. 30A is the same as FIG. 20A. FIG. 30B is the same as FIG. 20B. [Diagram 31]Figure 31A-C shows exemplary data correlating the number of CD11c+OPN+ microglia in human brains with the severity of Alzheimer's disease. (A) Middle frontal gyrus of AD patients and controls was stained for Iba-1 (microglia, red), CD11c (green) and OPN (cyan). (B) The percentage of CD11c+OPN+ microglia was significantly increased in brains of AD patients (CDR ≥ 1, n = 8) compared to MCI (CDR 0.5, n = 9) and control subjects (CDR 0, n = 5). Each dot represents the mean percentage of CD11c+OPN+ microglia relative to total Iba-1+ cells in 10-12 fields of view of each brain sample. HPF: high power field. ****p<0.0001 by one-way ANOVA with Tukey's multiple comparison test). (C) The percentage of CD11c+OPN+ microglia in the brain positively correlates with the CDR score in AD patients, MCI and controls. r=0.8383, p<0.0001 by Pearson's correlation analysis. Figures 31A-C are the same as Figures 20C-E, respectively. [Diagram 32] Figures 32A-B show exemplary data correlating brain OPN levels and the number of CD11c+OPN+ microglial cells with plaque scores in human brains of Alzheimer's disease patients. (A)(B) Brain OPN expression (r=0.4919, p=0.0043) and the percentage of CD11c+OPN+ microglia (r=0.8226, p<0.0001) positively correlate with plaque assessment by Pearson correlation analysis. Figure 32A is the same as Figure 20F. Figure 32B is the same as Figure 20G. [Diagram 33] 33A-B show an exemplary schematic (A) and data (B) for the generation and binding activity of Angiopep-2 conjugated monoclonal antibodies. (A) Preparation of AF488-labeled Angiopep-2 α-CD11b mAb conjugate (K: lysine). (B) After incubation with microglia from 5XFAD mice, Angiopep-2 conjugated and unconjugated α-CD11b mAb showed similar binding activity (AF488 CD11b+ microglia) and mean fluorescence intensity (MFI) (n=3). [Diagram 34] Exemplary data for Angiopep-2 conjugated anti-CD11B monoclonal antibody and its brain penetration in 5XFAD mice. Angiopep-2 conjugated α-CD11b mAb showed increased brain penetration (AF488+ microglia) in the brains of 5XFAD mice (n=3) as judged by the number of surface fluorescent CD11b+ microglia. (Two-tailed Student's t-test) ****p<0.0001. [Diagram 35] Exemplary data for anti-OPN monoclonal antibodies inhibiting microglial TNF-α production in microglial cells from 5XFAD mice are shown. In vitro study: Effect of anti-OPN mAb on microglial TNF-α production: Protocol: Microglia isolated from 9-month-old 5XFAD mice were cultured with different concentrations of anti-OPN mAb (MPIIIBl0(l)) for 24 hours, followed by analysis of TNF-α production using flow cytometry. Microglia incubated with isotype control (mouse IgG1) were used as negative control. [Diagram 36] FIG. 1 shows an exemplary schematic of OPN-dependent regulation of Aβ degradation and compaction by microglial cells. [Figure 37] An exemplary schematic of OPN mechanism of action is shown. A pathogenic OPN-producing CD11c+ microglia subset was identified that promotes AD development. The contribution of this CD11c+OPN+ microglia to AD pathology and cognitive impairment was defined. Therapeutics targeting OPN in preclinical AD models were developed to improve disease pathology and cognition. [Figure 38] 1 shows exemplary data of the effect of genetic deletion of OPN on Aβ degradation and plaque disposal in 5XFAD mice. [Figure 39] 1 shows exemplary data relating to OPN in Alzheimer's disease in 5XFAD mice and humans. [Diagram 40] 1 shows a schematic and data for one embodiment of the therapeutic compound disclosed herein. [Diagram 41] 1 shows an exemplary schematic diagram of an embodiment of the therapeutic compounds disclosed herein. [Diagram 42] Figure 42A-G shows that administration of anti-OPN mAb inhibits microglial proinflammatory responses and ameliorates Aβ plaque pathology. (A) Schematic of anti-OPN mAb administration. Anti-OPN mAb or isotype control (mouse IgG1) was intravenously injected (10 mg / kg) once a week into 5XFAD mice for 1 or 2 months starting at 6 months of age. Microglial proinflammatory responses and Aβ plaque pathology were analyzed after 1 or 2 months of treatment. (B) The percentages of CD11c+ microglia and TNF-α+CD11c+ microglia were determined by flow cytometry analysis. One month of treatment with anti-OPN mAb resulted in a modest reduction in CD11c+ microglia and a 35% reduction in TNF-α expression by CD11c+ microglia compared to age-matched 5XFAD mice treated with the same isotype IgG1 (n=3). Two months of treatment resulted in a 45% reduction in CD11c+ microglia and a 55% reduction in TNF-α production by CD11c+ microglia (n=3–4). (by two-tailed Student's t-test) **p<0.01, *p<0.05, ns: not significant. (C, D) Representative immunofluorescence images of 5XFAD mouse brains after 1 or 2 months of treatment with anti-OPN mAb or isotype control. Immunofluorescence after staining with 6E10 (red) and Thioflavin-S (green) is shown, highlighting the condensed and diffuse morphology of Aβ plaques. Scale bar=100 μm. (E-G) Quantification of total plaque area (6E10+), diffuse plaque area (6E10+Thio-S-) and plaque compactness index in 5XFAD mice after 1 or 2 months of treatment with anti-OPN mAb compared to isotype-treated mice (n=3). Compactness index is calculated as 6E10+Thio-S+ area / 6E10+ area (n=9 fields from 3 mice / group). *p<0.05 (by two-tailed Student's t-test), ns: not significant. All data are shown as mean ± sem. [Diagram 43]FIG. 43A-C shows that administration of cyclic RGD (Cilengitide) inhibits microglial pro-inflammatory responses. (A) Schematic of cyclic RGD (Cilengitide) administration. 5XFAD mice were intravenously injected (25 mg / kg) or intranasally delivered (10 mg / kg) once a week for 1 or 2 months starting at 6 months of age with Cilengitide or vehicle control (saline). Microglial pro-inflammatory responses were analyzed after 1 or 2 months of treatment. (B) Intranasal (IN) delivery of Cilengitide for 1 month resulted in a reduced prevalence of CD11c+ microglia and a 35% reduction in TNF-α production by CD11c+ microglia in 5XFAD mice compared to vehicle (saline) treated control mice (n=3). One month of IV injection of Cilengitide reduced the percentage of CD11c+ microglia by 50% and reduced TNF-α production by CD11c+ microglia by approximately 50% in 5XFAD mice (n=3). *p<0.5. (C) Intranasal (IN) delivery of Cilengitide for two months reduced the percentage of CD11c+ microglia by 35% and resulted in approximately 45% reduction in TNF-α expression by CD11c+ microglia. Two months of IV injection of Cilengitide reduced the percentage of CD11c+ microglia by 60% and reduced TNF-α production by CD11c+ microglia by 55% in 5XFAD mice compared to vehicle (saline)-treated control mice (n=3). *p<0.05, **p<0.01, ***p<0.001. [Diagram 44]FIG. 44A-C shows intranasal delivery of anti-CD11b monoclonal antibody (e.g., antibody not containing the conjugate-KK-amino acid bridge-AhX linker-angiopeptide-2 moiety). Intranasal delivery of the antibody (10 mg / kg body weight) to 6-month-old 5XFAD mice (n=4) was performed at time 0. The antibody was labeled with the fluorophore AF488. Observations were performed 3 hours after administration. The control was an intravenous injection of the same unconjugated anti-CD11b monoclonal antibody. (A) Anti-CD11b monoclonal antibody fluorescence in the brain (green) for intranasal administration vs. control intravenous administration. (B) Anti-CD11b monoclonal antibody fluorescence in the brain (green) for intranasal administration, along with immunofluorescence of anti-Iba-1 antibody (red). Iba-1 is a microglial marker. The merged immunofluorescence (yellow) confirms the binding specificity of anti-CD11b monoclonal antibody to microglia. (C) Data for calculation of anti-CD11b monoclonal antibody penetration into the brain in these experiments are shown as the number of fluorescent microglia per microscopic field (µm2 x 106) for anti-CD11b monoclonal antibody administered via the intranasal route compared to intravenous administration. The data show that intranasal administration of the antibody resulted in an approximately 10-fold increase in brain penetration compared to intravenous injection. [Diagram 45]FIG. 45A-C shows exemplary results related to OPN and inflammasome activation. (A) Activation of caspase-1 and its specificity. Intracellular caspase-1 activity was measured by bioluminescence assay in microglia from 9-month-old 5XFAD mice. Detection of specificity of caspase-1 activity was confirmed by selective caspase-1 inhibitor (Ac-YVAD-CHO, 1 μM). Bar graphs are representative results of three independent experiments. (B-C) show the effect of OPN-αVβ3 interaction on microglial caspase-1 activation (B) and IL-1β production (C). Activation of caspase-1 and IL1β by feeding rmOPN was blocked after feeding αVβ3 inhibitor to microglia from 9-month-old 5XFAD mice and OPN-KO.5XFAD mice (n=3). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant (one-way ANOVA with Bonferroni's multiple comparison test). UD not detectable. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention Microglial cells are a type of macrophage found in the central nervous system (CNS). Disclosed herein is a subset of microglial cells that are CD11c+ and produce osteopontin (OPN) in the brain. CD11c+ microglia contribute to the elimination of neuronal synapses by phagocytosing synaptic proteins early in development and can mediate proinflammatory responses during aging, an activity that declines in the absence of OPN. CD11c+OPN- microglial cells phagocytosed Aβ (approximately 60%), expressed high levels of TREM2, produced negligible levels of TNF-α, and may be protective. In contrast, only a small proportion of CD11c+OPN+ microglial cells ingest Aβ, these cells express low levels of TREM2, and many produce TNF-α. OPN production by these cells may reflect enhanced proinflammatory responses in activated lysosomes and impaired TREM2-dependent Aβ plaque fixation. CD11c+OPN+ microglial cells may represent a pathogenic microglial subset and contribute to the symptoms of Alzheimer's disease (AD). These cells may be targets for therapeutic approaches.

[0023] The transport of substances (e.g., therapeutic agents) from the blood to the brain can be regulated by capillary endothelial cells in the brain, called the blood-brain barrier (BBB). The transport of substances across the BBB is limited and selective. Molecules with certain properties can cross the BBB by passive diffusion or active / facilitated transport and transcytosis. However, many substances are excluded from transport across the BBB. In some instances, molecules that do not normally cross the BBB can be crossed using various methods.

[0024] Disclosed herein are approaches for transporting compounds across the blood-brain barrier (BBB). The compounds can include a payload or active agent, including therapeutic agents, cytotoxic agents, imaging agents, and the like. The compounds generally include a moiety for delivering the payload or active agent across the BBB. In some embodiments, the active agent can be conjugated to the moiety. In some embodiments, the payload or active agent can be a therapeutic agent for treating a brain disease or affliction. In some embodiments, the therapeutic agent can be an antibody or protein-based therapeutic agent.

[0025] In some embodiments, the moiety for delivering the payload / active agent across the BBB can include a carrier agent. The moiety can include a bridging moiety, which can be an amino acid bridging moiety (e.g., arginine, histidine and / or lysine residue), which can be positively charged. The moiety can include a linker, which can be a peptide linker, which can be a flexible linker. The moiety can include one or more of a carrier agent, a bridging moiety, a linker, and combinations thereof.

[0026] In some embodiments, brain diseases or afflictions can be treated by the compounds and methods for transporting therapeutic agents across the BBB. These diseases can include inflammatory diseases or cancer. In some embodiments, osteopontin (OPN)-mediated neuroinflammatory diseases, such as multiple sclerosis (MS) and Alzheimer's disease (AD), can be treated.

[0027] In some embodiments, compounds and methods for transporting therapeutic agents across the BBB can be used to target microglial cells that have OPN (e.g., CD11c+OPN+ microglial cells). In some embodiments, the therapeutic agent can be an antibody specific for CD11c, OPN, or CD11c and OPN. These antibodies can target CD11c+OPN+ microglial cells. In some embodiments, the therapeutic agent can be an antibody specific for OPN.

[0028] Aspects of the invention relate to artificial, brain-penetrant therapeutic compounds. Aspects of the invention further relate to methods of treating neurological disorders, such as Alzheimer's disease and dementia.

[0029] A detailed description of one or more preferred embodiments is provided herein. However, it is understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be interpreted as limiting, but as a basis for the claims and as a representative basis for teaching a person skilled in the art to use the present invention in any suitable manner.

[0030] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0031] Whenever any of the phrases "for example," "such as," "including," and the like are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is accompanying. Similarly, "an example," "exemplary," and the like are understood to be non-limiting.

[0032] The term "substantially" permits deviations from the descriptors that do not adversely affect the intended purpose. It is understood that the descriptors are modified by the term "substantially" even if the word "substantially" is not expressly recited.

[0033] Terms such as "comprising," "including," "having," and "involving" (and similarly "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the general U.S. patent law definition of "comprising," and therefore is to be construed as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they are to be understood as "one or more," unless such an interpretation is insignificant in the context.

[0034] As used herein, the term "about" is used herein to mean approximately, roughly, approximately, or within the region. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the set forth value by a variance of up to 20 percent (higher or lower).

[0035] An aspect of the present invention relates to a method for treating a neurological disorder. For example, the method comprises administering a therapeutically effective amount of the composition described herein to a subject. In another example, the neurological disorder can include a neurodegenerative disease. In another example, the neurodegenerative disease can include Alzheimer's disease.

[0036] As used herein, "treatment" and "treating" can refer to the management and care of a subject in any manner in which one or more of the symptoms of a disease or disorder are improved or otherwise beneficially altered for the purpose of combating the condition, disease, or disorder. The term "treat" or "treatment" can also refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) an undesirable physiological change or disorder. The term can include a full range of treatments for a given condition suffered by a patient, such as administration of an active compound for the purpose of reducing or alleviating symptoms or complications; slowing the progression of the condition, disease, or disorder; curing or eliminating the condition, disease, or disorder; and / or preventing the condition, disease, or disorder, where "preventing" or "prevention" can refer to the management and care of a patient for the purpose of preventing the onset of the condition, disease, or disorder, and can include administration of an active compound to prevent or reduce the risk of onset of the symptoms or complications. "Treatment" can also refer to prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in need of prevention of a condition or disorder. The subject or patient to be treated may be a mammal, such as a human. Treatment also includes any pharmaceutical use of the compositions herein, such as use to treat a disease as provided herein.

[0037] In embodiments, a method for preventing or treating a neurological disorder in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a composition described herein.

[0038] The term "neurological disorder" refers to any condition of the central or peripheral nervous system of a mammal. The term "neurological disorder" includes neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis), neuropsychiatric disorders (e.g., schizophrenia and anxiety disorders, e.g., generalized anxiety disorder). Exemplary neurological disorders include MLS (cerebellar ataxia), Huntington's disease, Down's syndrome, multi-infectious dementia, status epilepticus, contusion (e.g., spinal cord injury and head injury), viral infection-induced neurodegeneration (e.g., AIDS, encephalopathy), epilepsy, benign forgetting, closed head injury, sleep disorder, depression (e.g., bipolar disorder), dementia, movement disorder, psychosis, alcoholism, post-traumatic stress disorder, and the like. "Neurological disorder" also includes any condition associated with the disorder. For example, a method of treating a neurodegenerative disorder includes a method of treating memory loss and / or cognitive loss associated with a neurodegenerative disorder. Exemplary methods also include treating or preventing loss of neuronal function characteristic of a neurodegenerative disorder. "Neurological disorder" also includes any disease or condition (e.g., cardiovascular disease) that is at least partially implicated in monoamine (e.g., norepinephrine) signaling pathways.

[0039] As used herein, "Alzheimer's disease," "Alzheimer's disease," or "AD" refers to a disease in which cognitive function is gradually impaired over time, including symptomatic pre-dementia manifested by mild cognitive impairment (MCI), and dementia stage in which there is significant impairment in social or occupational functioning.

[0040] As used herein, "diagnosis" or "prognosis" refers to the use of information (e.g., data from genetic or other molecular testing, biological or chemical information from a biological sample, signs and symptoms, physical exam findings, cognitive performance results, etc.) to predict the most likely outcome, time frame, and / or response to a given treatment for a given disease, disorder, or condition, based on comparison with multiple individuals who share a common nucleotide sequence, symptoms, signs, family history, or other data relevant to a consideration of the patient's health status, or based on identification of a subject's affliction with, for example, Mild Cognitive Impairment (MCI) (e.g., Alzheimer's type cognitive impairment).

[0041] As used interchangeably herein, a "subject," "individual," or "patient" can refer to a vertebrate such as a mammal, e.g., a human. Mammals include, but are not limited to, rats, monkeys, humans, farm animals, sport animals, and pets. The term "pets" includes dogs, cats, guinea pigs, mice, rats, rabbits, ferrets, and the like. The term farm animals includes horses, sheep, goats, chickens, pigs, cows, donkeys, llamas, alpacas, turkeys, and the like.

[0042] The term "administration" can refer to the introduction of a pharmaceutical composition or formulation described herein into a subject. One of the routes of administration of the composition is intravenous administration. However, any route of administration can be used, such as oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, intranasal, introduction into cerebrospinal fluid, or instillation into a body compartment. Without wishing to be bound by theory, in some embodiments, administration of an active agent that includes a moiety for delivery of the active agent through the blood-brain barrier can result in the active agent crossing the blood-brain barrier and entering the brain.

[0043] In some embodiments, nasal or intranasal administration can be used to administer and deliver the active agent to the central nervous system (e.g., the brain). In some embodiments, nasal or intraductal delivery of the active agent can use a liquid nasal spray. Without wishing to be bound by theory, intranasal administration of the active agent can result in olfactory transmission of the active agent to the brain. In some embodiments, the nerve cells of the olfactory epithelium, which are present in the nasal cavity and project into the olfactory bulb of the brain, can provide a connection between the brain and the external environment. In some embodiments, the intranasally administered active agent can travel along the olfactory nerve cells and enter the brain, usually bypassing the blood-brain barrier. In some embodiments, the active agent administered via the intranasal route may not include a moiety for delivery of the active agent across the blood-brain barrier.

[0044] In some embodiments, intranasal administration can result in the active agent entering the bloodstream. The active agent can then cross the blood-brain barrier and enter the brain. In some embodiments, these active agents can include a moiety or moieties to facilitate delivery to the brain. The active agent can enter the brain via different mechanisms.

[0045] The term "therapeutically effective amount" can refer to that amount of an embodiment of a compound or pharmaceutical composition being administered that will alleviate to some extent one or more symptoms of the disease or condition being treated and / or prevent to some extent one or more symptoms of a condition or disease that the treated subject develops or is at risk of developing. In one embodiment, a therapeutically effective amount can refer to the amount necessary to treat at least one pathological effect resulting from the presence of a neurological disorder, such as Alzheimer's disease, or a neurological condition in a human or animal subject.

[0046] As used herein, "active agent" or "payload" refers to a substance, such as a therapeutic agent, a cytotoxic agent, an imaging agent, etc., that is part of a compound disclosed herein that is transported across the blood-brain barrier (BBB). A "therapeutic agent" refers to a substance that, when administered to a subject, can treat a disease or condition in the subject.

[0047] As used herein, "adsorptive-mediated transcytosis" or "AMT" refers to the adsorption of a substance at the luminal surface and exocytosis of a substance at the abluminal surface of an endothelial cell. This transport across the endothelial cell usually involves vesicles.

[0048] As used herein, "antibody" refers to one or more molecules that bind to an antigen. As used herein, "antibody" generally refers to any type of antibody, fragment, and / or derivative. Antibodies include polyclonal and monoclonal antibodies of any appropriate isotype or isotype subclass. As used herein, an antibody may refer to, but is not limited to, Fab, F(ab')2, Fab' single chain antibody, Fv, single chain, monospecific antibody, bispecific antibody, trispecific antibody, multivalent antibody, chimeric antibody, dog-human chimeric antibody, chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, shark antibody, nanobody (e.g., an antibody consisting of a single monomeric variable domain), camelid antibody (e.g., camelid) microbody, intrabody (e.g., intracellular antibody), and / or defucosylated antibody and / or derivatives thereof. Antibody mimetics are also provided. In some embodiments, the antibody disclosed herein is an active agent that is part of a compound disclosed herein that can cross the blood-brain barrier.

[0049] As used herein, "blood-brain barrier" or "BBB" refers to the block of cells between the blood and substances in the blood and the brain. Generally, the BBB is composed of endothelial cells and other cells.

[0050] As used herein, a "bridge" may refer to a portion of the compound or a moiety disclosed herein. A bridge may connect other components of the compound or moiety. In some embodiments, a bridge is an amino acid bridge. An amino acid bridge may refer to one or more amino acids that form a peptide bond and connect at least two components of the compound or moiety, such as a therapeutic agent, a carrier agent, a flexible linker, etc. In embodiments, a bridge may cross the blood-brain barrier. In embodiments, a bridge may cross the blood-brain barrier by adsorptive-mediated transcytosis (AMT).

[0051] As used herein, a "carrier agent" can refer to a first substance that is facilitated by a second substance to pass through the blood-brain barrier, and the second substance alone does not normally pass through the BBB. The carrier agent is part of a compound or moiety disclosed herein. In some embodiments, the carrier agent can pass through the blood-brain barrier. In embodiments, the carrier agent can pass through the blood-brain barrier through specific interaction with a corresponding receptor expressed on the cells of the blood-brain barrier. In some embodiments, the carrier agent passes through the blood-brain barrier by receptor-dependent transcytosis (RDT).

[0052] As used herein, a "compound" refers to one or more active agents linked to one or more moieties. In general, the active agent and the moieties (e.g., carrier agents, bridging moieties, linkers, and combinations thereof) comprise a compound described herein. In some embodiments, the active agent can be conjugated to the moiety. When the active agent is conjugated to the moiety, the "compound" can include the active agent, the moiety, and the conjugate.

[0053] As used herein, "conjugated to" may refer to a chemical bond between one substance and another. Herein, a payload or active agent is generally conjugated to a moiety that crosses the blood-brain barrier. "Conjugate" may refer to a chemical bond that connects an active agent and a moiety.

[0054] As used herein, a "linker" is part of certain embodiments of the compounds or moieties disclosed herein. In some embodiments, a "flexible linker" refers to an amino acid that, when added to a compound or moiety of a protein or polypeptide, can increase the flexibility of the protein or polypeptide.

[0055] As used herein, a "moiety" refers to one or more substances attached to an active agent that facilitates passage of the active agent through the BBB. In various embodiments, as described herein, a moiety can include a carrier agent, a carrier agent plus a bridging moiety, a carrier agent plus a linker, or a carrier agent plus a bridging moiety and a linker.

[0056] As used herein, "microglial cells" refers to a population of macrophages derived from the central nervous system (CNS). In the brain, these cells can eliminate damaged and infected cells.

[0057] As used herein, "osteopontin" refers to a secreted phosphorylated protein encoded by the SPP1 gene in humans and the Spp1 gene in mice.

[0058] As used herein, "receptor-mediated transcytosis" or "RDT" may refer to the binding of a substance to a receptor on the luminal surface of an endothelial cell and exocytosis of the substance at the abluminal surface. This transport across the endothelial cell usually involves vesicles.

[0059] As used herein, the term "5XFAD mouse" refers to a mouse model that reproduces the amyloid pathology of Alzheimer's disease (Oakley, Holly, et al. "Intraneuronal β-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation." Journal of Neuroscience 26.40 (2006): 10129-10140).

[0060] Osteopontin (OPN) antibody Described herein is a unique recombinant monoclonal OPN antibody. "Recombinant" in reference to a polypeptide (such as an antibody) or polynucleotide refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which can be made by combining polynucleotides or polypeptides that do not normally go together. As used herein, "polypeptide" can encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids can refer to a "polypeptide" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but need not necessarily be translated from a specific nucleic acid sequence. It can be generated in any manner, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, delete, or replace a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the modification replaces an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.Such conservatively modified variants of the anti-OPN antibodies disclosed herein can exhibit increased cross-reactivity to OPN compared to an unmodified OPN antibody.

[0061] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art as: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0062] The OPN antibodies described herein bind to osteopontin. In one embodiment, the OPN antibodies have high affinity and high specificity for osteopontin. In some embodiments, the osteopontin antibody is monoclonal antibody MPIIIB10(1), available from the Developmental Studies Hybridoma Bank (https: / / dshb.biology.uiowa.edu / MPIIIB10-1), which is reactive to at least human, mouse, rat, dog, and chicken osteopontin. Other anti-osteopontin monoclonal antibodies include at least 7C5H12, 2F10, OTI5E4, 4H7, OTI2F2, OSP / 4589, OT16C12, OTI6A12, OTI3C4, or 1E10 (https: / / www.thermofisher.com / antibody / primary / target / osteopontin), OPN46 (https: / / www.sigmaaldrich.com / US / en / product / sigma / sab4200018), clone 53 (https: / / www.enzolifesciences.com / ADI-905-629 / osteopontin-monoclonal-antibody-53 / ), etc. Other anti-osteopontin antibodies can be used.

[0063] Some embodiments also feature antibodies that have a certain percentage of identity or similarity to the amino acid or nucleotide sequences of the anti-OPN antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing the position of each sequence, which may be aligned for comparison purposes. If a position of the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity when compared to a specific region or the entire length of any one of the anti-OPN antibodies described herein. For example, an antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity when compared to a specific region or the entire length of any one of the anti-OPN antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Sequence comparison and / or alignment can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity of the nucleic acids and proteins of the invention.

[0064] An aspect of the invention provides an isolated antibody specific for OPN. When used herein with respect to a cell, a nucleic acid such as DNA or RNA, the term "isolated" refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material or medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. For example, an "isolated nucleic acid" can include a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated" can also refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. An isolated polypeptide can include both purified and recombinant polypeptides.

[0065] As used herein, an "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody and any antigen-binding fragment, or single chain thereof. For example, an "antibody" can include any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the heavy or light chain variable region, the heavy or light chain constant region, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunoglobulin (Ig) molecules, i.e., immunologically active portions of molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides.

[0066] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to (ab’)2 , F (ab)2 , F ab ', F ab "Antibody fragment" refers to a portion of an antibody, such as a Fv, scFv, etc. Antibody fragments, regardless of structure, bind to the same antigen recognized by the complete antibody. The term "antibody fragment" can include aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab', F(ab')2, Fd, Fvs, single chain Fv (scFv), single chain antibodies, dAbs (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0067] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L Single chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed from gene fusions containing VH and VL encoding genes linked by a peptide-encoding linker. (Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some embodiments, the domains are linked by a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility and serine or threonine for solubility, and may be rich in the VH:VL heterodimers. H N-terminus of V LThe C-terminus of the scFv molecule may be linked to the C-terminus of the antibody V region or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Many methods have been described for identifying chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from the antibody V region into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,892,019, 5,132,405, and 4,946,778 (each of which is incorporated herein by reference in its entirety).

[0068] Antibody molecules obtained from humans are classified into five classes of immunoglobulins, IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chain present in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within them (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0069] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0070] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL, and CH1, CH2 or CH3) of the light and heavy chains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions", are interposed between more conserved adjacent sections known as "framework regions" or "FRs". Thus, the term "FR" can refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface that is complementary to the three dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs."

[0071] The six CDRs present in each antigen-binding domain are short non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show less intermolecular variation. The framework regions adopt a predominantly beta-sheet conformation, and the CDRs form loops that connect and in some cases form part of the beta-sheet structure. The framework regions function to form a scaffold for positioning the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the antigen in the immune response, facilitating non-covalent binding of the antibody to its cognate epitope. The amino acids which comprise the CDRs and framework regions, respectively, have been previously identified (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)) and can be readily identified by one of skill in the art for a heavy or light chain variable region.

[0072] In the event that there is more than one definition for a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless specifically and explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. This region is described by Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDR by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is intended that the application of either definition to refer to the CDRs of an antibody or variants thereof is within the scope of the term as defined and used herein. The appropriate amino acid residues encompassing the CDRs defined by each of the above cited references are set forth in the following table for comparison. The exact residue numbers which comprise a given CDR will vary depending on the sequence and size of the CDR, and one of skill in the art can routinely determine which residues make up a given CDR given the variable region amino acid sequence of an antibody. TIFF2025508682000002.tif47128

[0073] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on other experimental data of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0074] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins about the 15th residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids, and ends with the sequence WGXG (X is any amino acid). CDR-L1 begins about residue 24 (i.e., after the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins about the 16th residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0075] As used herein, the terms "nanobody" and "isolated VHH domain" can be used interchangeably and refer to single domain antibody fragments from camelids. "Nanobody" refers to the smallest antigen-binding fragment or single variable domain ("VHH") derived from a naturally occurring heavy chain antibody. Nanobodies are derived from heavy chain only antibodies and are found in camelids. In the "camelid" family, immunoglobulins that lack light polypeptide chains are found. "Camelids" include Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., alpacas, Lama glama, Lama guanicoe, and Lama vicugna). Nanobodies with low specificity bind to several different epitopes (or polypeptide regions) via a single antigen-binding site or binding domain, whereas Nanobodies with high specificity bind to one or a few epitopes (or polypeptide regions) via a single antigen-binding site or binding domain.

[0076] It should be noted that the term "Nanobody", as used herein in its broadest sense, is not limited to a particular biological source or a particular method of preparation. For example, the Nanobodies herein can generally be obtained (1) by isolating the VHH domain of a naturally occurring heavy chain antibody, (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain, (3) by "humanization" of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain, (4) by "camelization" of a naturally occurring VH domain from any animal species, for example from a mammalian species such as human, or by expression of a nucleic acid encoding such a camelized VH domain, (5) by "camelization" of "domain antibodies" or "Dabs" as reported in the art or by expression of a nucleic acid encoding such a camelized VH domain, (6) by using synthetic or semi-synthetic techniques for the preparation of proteins, polypeptides or other amino acid sequences known per se, (7) by preparing a nucleic acid encoding a Nanobody using nucleic acid synthesis techniques known per se and then expressing the nucleic acid thus obtained, and / or (8) by any combination of one or more of the above.

[0077] The term "monobody" as used herein refers to an antigen-binding molecule that has a heavy chain variable domain and no light chain variable domain. Monobodies can bind antigen in the absence of light chains and have three CDR regions called CDRH1, CDRH2 and CDRH3. Heavy chain IgG monobodies have two heavy chain antigen-binding molecules linked by disulfide bonds. The heavy chain variable domain contains one or more CDR regions, e.g., the CDRH3 region. "VhH" or "VHH" refers to the variable domain of a heavy chain antibody such as a monobody. "Camelidae monobody" or "Camelidae VHH" refers to a monobody or antigen-binding portion thereof obtained from an animal of Camelidae origin, including animals with feet with two toes and leathery soles.

[0078] The term "DARPin" (artificial ankyrin repeat protein) refers to an antibody mimetic protein with high specificity and high binding affinity to a target protein, prepared by genetic engineering. DARPins are derived from natural ankyrin proteins and have a structure containing at least two ankyrin repeat motifs, for example, a structure containing at least three, four or five ankyrin repeat motifs. DARPins can have any suitable molecular weight depending on the number of repeat motifs. DARPins comprise a core portion that provides a structure and a target binding portion that is present outside the core and binds to the target. The structural core comprises a conserved amino acid sequence, and the target binding portion comprises a different amino acid sequence depending on the target. DARPins have target specificity similar to antibodies. Thus, new forms of bispecific chimeric proteins are obtained by binding DARPins to antibodies or antibody fragments, such as IgG (for example, IgG1, IgG2, IgG3 or IgG4) antibodies or scFv-Fc antibody fragments.

[0079] As used herein, the term "affibody" refers to a protein engineered to bind to a target protein or peptide with high affinity, mimicking a monoclonal antibody, and thus a member of the family of antibody mimetics. Affibodies are composed of a three-helix bundle domain derived from the IgG-binding domain of Staphylococcus aureus protein A. The protein domain consists of a 58 amino acid sequence, with 13 randomized amino acids resulting in a range of affibody variants. Despite being much smaller than antibodies (affibodies weigh about 6 kDa, whereas antibodies weigh about 150 kDa), affibody molecules act like antibodies, since their binding site is roughly equivalent in surface area to that of an antibody.

[0080] As used herein, the term "epitope" can include any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of the complementarity determining regions (CDRs), combine to form the variable regions that define a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of a Y. Epitope determinants may consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by the three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains.

[0081] In some embodiments, the antibody can be directed against osteopontin (OPN). In humans, osteopontin is encoded by the SPP1 gene (secreted phosphoprotein 1). The mouse ortholog is Spp1. In some embodiments, the antibody can be directed against human osteopontin (OPN). Osteopontin is generally secreted. Intracellular forms of osteopontin can exist. For example, variants of osteopontin can exist in certain cancer cells.

[0082] In some embodiments, human osteopontin is encoded by a gene having NCBI GenBank Gene ID 6696 (SPP1, secreted phosphoprotein 1). In some embodiments, mouse osteopontin is encoded by a gene having NCBI GenBank Gene ID 20750 (Spp1, secreted phosphoprotein 1). In some embodiments, osteopontin (from rat) has NCBI GenBank reference number: AAA41765.1 (317 amino acid residues long) and is set forth in SEQ ID NO:2: MRLAVVCFCLFGLASCLPKVAEFGSSEEKAHYSKHSDAVATWLKPDPSQKQNLLAPQNSVSSEETDDFKQETLPSNSNESHDHMDDDDDDDDDGHAESEDSVNSDESDESHHSDESDESFTASTQADVLTPIAPTVDVPDGRGDSLAYGLRSKSRS FPVSDEQYPDATDEDLTSRMKSQESDEAIKVIPVAQRLSVPSDQDSNGKTSHESSQLDEPSVETHSLEQSKEYKQRASHESTEQSDAIDSAEKPDAIDSAERSDAIDSQASSKASLEHQSHEFHSHEDKLVLDPKSKEDDRYLKFRISHELESSSSEVN The amino acid sequence of

[0083] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ), and K dA smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on-rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentrations and actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay, e.g., a radioligand binding assay or similar assay known to those of skill in the art, e.g., BIAcore or Octet (BLI). D ) is ≦1 μM, ≦10 μm, ≦10 nM, ≦10 pM, or ≦100 pM to about 1 pM. For example, in some embodiments, D is between about 1E-12M and about 1E-11M. D In some embodiments, K D is between about 1E-11M and about 1E-10M. D In some embodiments, K D is between about 1E-10M and about 1E-9M. D In some embodiments, K D is between about 1E-9M and about 1E-8M. D In some embodiments, K D is between about 1E-8M and about 1E-7M D In some embodiments, K D is between about 1E-7M and about 1E-6M DFor example, in some embodiments, K D is about 1E-12M, and in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, and in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, and in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, and in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, and in other embodiments, K D is about 3E-12M. In some embodiments, K D is about 6E-11M. "Specifically binds" or "having specificity" can refer to an antibody that binds to an epitope through the antigen-binding domain of the antibody, where the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope.

[0084] For example, the OPN antibody can be monovalent or bivalent and can comprise a single chain or two chains. Functionally, the binding affinity of the OPN antibody is greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of the OPN antibody is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 - M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It's M.

[0085] OPN protein, or a derivative, fragment, analog, homolog, or ortholog thereof, can be used as an immunogen in the generation of antibodies that immunologically specifically bind to these protein components, such as amino acid residues comprising SEQ ID NO: 2. Proteoliposome-bound OPN protein, or a derivative, fragment, analog, homolog, or ortholog thereof, can be used as an immunogen in the generation of antibodies that immunospecifically bind to these protein components.

[0086] One of ordinary skill in the art will recognize that one can determine, without undue experimentation, whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the former prevents the latter from binding to OPN. If the human monoclonal antibody being tested competes with a human monoclonal antibody of the invention, e.g., as indicated by reduced binding by a human monoclonal antibody of the invention, then the two monoclonal antibodies may bind to the same or closely related epitopes.

[0087] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the OPN protein with which it is normally reactive, then add the human monoclonal antibody to be tested and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to OPN. If the human monoclonal antibody to be tested is inhibited, then it likely has the same, or a functionally equivalent, epitopic specificity as the monoclonal antibody of the present invention. Screening of the human monoclonal antibodies of the present invention can also be performed using OPN to determine whether the monoclonal antibody to be tested can neutralize OPN.

[0088] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0089] Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0090] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. For example, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a given epitope of an antigen characterized by a unique binding affinity for it.

[0091] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0092] Activator The compounds disclosed herein generally include a substance desired to be delivered to the brain and are referred to as an active agent or payload. In some embodiments, the active agent may include a therapeutic agent, a drug, a cytotoxic agent, an imaging agent, etc. In some embodiments, the active agent may include a protein. In some embodiments, the active agent may include an antibody or a protein-based therapeutic agent.

[0093] The present invention is also directed to immunoconjugates, including antibodies conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or to a radioisotope (i.e., a radioconjugate).

[0094] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitgellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0095] Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies (see WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0096] Those of skill in the art will appreciate that a wide variety of moieties may be attached to the resulting antibodies or other molecules of the invention. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), incorporated herein by reference in its entirety.

[0097] The binding can be achieved by any chemical reaction that will link the two molecules, so long as the antibody and the other moiety retain their respective activities. The binding can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, complex formation. In one embodiment, the binding is a covalent bond. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of an external cross-linking molecule. Many bivalent or multivalent binding agents are useful for linking protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative binding agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of binding agents known in the art, but rather is illustrative of the more common binding agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982), and Vitetta et al., Science 238:1098 (1987).) Non-limiting examples of linkers are described in the literature. (For example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies via oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamide)]hexanoate (Pierce Chem. Co., Cat. Co., Cat. #2165-G), and (v) sulfo-NHS (-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0098] The linkers described herein contain components with different attributes, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT can contain sterically hindered disulfide bonds to form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds, because they are cleaved in vitro, resulting in fewer available conjugates. For example, sulfo-NHS can increase the stability of carbodiimide coupling. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0099] In some embodiments, the active agent can include an antibody or other agent (e.g., an anti-osteopontin antibody) that targets a particular cell type associated with the disease or affliction being treated. In some embodiments, the active agent can target cancer cells or cells associated with an autoimmune disorder. In some examples, the active agent can target microglial cells. Microglial cells can be CD11c+OPN+ cells. These cells can be found, for example, in people with Alzheimer's disease. In some embodiments, the active agent can be an antibody specific for osteopontin (OPN) or CD11.

[0100] In some embodiments, the active agent can include an integrin inhibitor. In some embodiments, the integrin inhibitor can include an αVβ3 inhibitor. The αVβ3 inhibitor can include, for example, antibodies such as LM609 / Avastin, CNTO 95, c7E3, and 17E6. The αVβ3 inhibitor can include, for example, antagonists such as disintegrins, peptides, and non-peptide molecules (e.g., peptidomimetics, siRNA).

[0101] In some embodiments, the αVβ3 inhibitor can be a cyclic RGD-containing small molecule or a derivative thereof. In some embodiments, it can be Cilengitide or a derivative thereof. Cilengitide is a head-to-tail cyclized RGD (arg-gly-asp)-containing pentapeptide that can bind to integrins αVβ3 and αVβ5. Cilengitide is designed to compete with the RGD peptide sequence that normally regulates integrin-ligand binding. For example, Cilengitide blocks ligation of αVβ3 and αVβ5 integrins to matrix proteins such as vitronectin, fibronectin, fibrinogen, von Willebrand factor, and osteopontin.

[0102] In some embodiments, the αVβ3 inhibitor can be administered as part of a therapeutic regime in which an anti-osteopontin antibody is administered. In some embodiments, the αVβ3 inhibitor can be administered in combination with an anti-osteopontin antibody. The αVβ3 inhibitor can be conjugated to a crosslinking moiety, a linker, a carrier agent, or a combination thereof. The αVβ3 inhibitor may not be conjugated to a crosslinking moiety, a linker, a carrier agent, or a combination thereof.

[0103] In the present disclosure, the active agent is generally bound to one or more moieties that allow the active agent to cross the BBB. The active agent plus one or more moieties is referred to as the "compound" of the present disclosure that crosses the BBB and is delivered to the brain. However, in some embodiments, the active agent may not be bound to one or more moieties that allow the active agent to cross the BBB.

[0104] Carrier Agent The carrier agent may be a moiety attached to the active agent or may be part of a moiety or moieties attached to the active agent. In some embodiments, the carrier agent may be attached directly to the active agent. In some embodiments, the carrier agent may be attached to the active agent via a conjugate. In some embodiments, the carrier agent may be attached to a crosslinker or flexible linker that is attached directly to the active agent or via a conjugate.

[0105] In some embodiments, the carrier agent can facilitate transport of the moiety or the compound containing the active agent and the moiety across the BBB, hi some embodiments, these substances can cross the BBB via receptor-mediated transcytosis (RDT).

[0106] In some examples, the carrier agent bound to the active agent can cause the active agent to pass through the BBB. In some examples, the first carrier agent may not be able to pass the BBB by itself, but can be combined with one or more second carrier agents or other molecules (e.g., other molecules such as bridging moieties and / or flexible linkers) to allow the active agent to pass through the BBB. In some examples, a single carrier agent can only allow a low level of the active agent to pass through the BBB, but can be increased by combining the same or different carrier agents.

[0107] In some embodiments, the carrier agent can be a peptide. In some embodiments, the carrier agent that is a peptide can mediate the passage of the active agent through the blood-brain barrier through the interaction of the peptide with its receptor on the blood-brain barrier. Angiopep-2 (TFFYGGSRGKRNNFKTEEY; SEQ ID NO: 1) is such a peptide. In some embodiments, the peptide can be a cell-penetrating peptide (CPP). Such a CPP can be less than 20 amino acids in length in some embodiments and can include amino acids with a positive charge. CPPs can pass through the cell membrane bilayer through interaction with the negatively charged cell membrane.

[0108] In embodiments, the peptide carrier agent may include D-Lys6-LHRH (SEQ ID NO:3), Angiopep-2, CNGRCG (SEQ ID NO:4), PGA, LHRH (SEQ ID NO:5), DRDDS (spacer; SEQ ID NO:6), D-γ-E-γ-E-γ-EE (masking moiety; SEQ ID NO:7), GSH, HSTPSSP (SEQ ID NO:8), DSSLFAL (SEQ ID NO:9) and others (Jafari, Behzad, et al. "Peptide-mediated drug delivery across the blood-brain barrier for targeting brain tumors." Expert opinion on drug delivery 16.6 (2019): 583-605.). In some embodiments, the carrier agent comprises Angiopep-2. Angiopep-2 may have the amino acid sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO:1) or a sequence at least 90% identical to SEQ ID NO:1.

[0109] In some embodiments, peptide carrier agents can include YGRKKRRQRRRPPQQ (TAT; SEQ ID NO: 10), LLIILRRRIRKQAHAHSK (pVEC; SEQ ID NO: 11), RRLSYSRRRF (SynB3; SEQ ID NO: 12), and the like.

[0110] In some embodiments, the carrier agent can be a positively charged, amphipathic peptide of up to 20 or 30 amino acids, known as a cell-penetrating peptide (Gao, Huile, et al. "Angiopep-2 and activatable cell-penetrating peptide dual-functionalized nanoparticles for systemic glioma-targeting delivery." Molecular pharmaceutics 11.8(2014):2755-2763.).

[0111] In some embodiments, the carrier agent may be a cell targeting peptide (Mousavizadeh, Ali, et al. "Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: A systematic review." Colloids and Surfaces B: Biointerfaces 158(2017):507-517.).

[0112] In general, any carrier agent can be used in the compositions and methods described herein, in some embodiments, any peptide carrier agent can be used in the compositions and methods described herein.

[0113] Bridge part The cross-linking moiety may be a moiety attached to the active agent or may be part of a moiety or moieties attached to the active agent. In some embodiments, the cross-linking moiety may be attached directly to the active agent. In some embodiments, the cross-linking moiety may be attached to the active agent via a conjugate. In some embodiments, the cross-linking moiety may be attached to a flexible linker or carrier agent that is attached directly to the active agent or via a conjugate. In some embodiments, the cross-linking moiety may be disposed between the active agent and the carrier agent.

[0114] In some embodiments, the crosslinker can facilitate transport of the moiety or a compound containing the active agent and the moiety across the BBB, hi some embodiments, these agents can cross the BBB via adsorptive-mediated transcytosis (AMT).

[0115] In general, the crosslinking moiety can be designed to enhance the ability of the moiety and attached active agent to be delivered across the BBB to the brain, in some embodiments, the crosslinking moiety can be designed to alter the charge of the compound containing the active agent (e.g., the active agent + moiety).

[0116] In some embodiments, the bridge can include amino acids in the form of peptides or polypeptides. The bridge can include amino acids that change the charge of the compound containing the active agent. In some embodiments, the amino acids that make up the bridge can be selected to increase or decrease the isoelectric point (pI) of the compound containing the active agent. In some embodiments, the amino acids of the bridge can be selected to increase the pI of the compound containing the active agent. In some embodiments, the amino acids of the bridge can be selected to increase the pI of the compound containing the active agent to an alkaline level. These amino acids may be positively charged (e.g., arginine, lysine, histidine). In some embodiments, the amino acid bridge can include 2, 3, 4, 5, 6 or more consecutive positively charged amino acids. In some embodiments, the bridge can include 2, 3, 4, 5, 6 or more consecutive lysine residues. In some embodiments, the bridge can include two consecutive lysine residues.

[0117] Increasing the pI of the compound can facilitate the transport of the moiety or the compound containing the active agent and the moiety through the BBB. In some embodiments, this transport can occur via adsorptive-mediated transcytosis (AMT). The increased positive charge of the compound can enhance the interaction between the compound and the surface of endothelial cells that make up the BBB. In general, the surface of these endothelial cells can be negatively charged.

[0118] Linker The linker may be a moiety attached to the active agent or may be part of a moiety or moieties attached to the active agent. In some embodiments, the linker may be attached directly to the active agent. In some embodiments, the linker may be attached to the active agent via a conjugate. In some embodiments, the linker may be attached to a carrier agent and / or a cross-linking moiety that is attached directly or via a conjugate to the active agent. In some embodiments, the linker may be disposed between the active agent and the carrier agent.

[0119] In general, linkers can be designed to increase the flexibility of the moiety and / or the compound that includes the moiety and the active agent. In some instances, this flexibility can facilitate solvation of the moiety or compound. In some instances, this flexibility can reduce aggregation of the moiety or compound.

[0120] In some embodiments, a linker may refer to an amino acid or peptide spacer that separates multiple domains (e.g., active agent, carrier agent) within a compound (e.g., a protein or polypeptide) that includes an active agent and a moiety.

[0121] In some embodiments, the linker can be a flexible linker. A flexible linker may refer to a linker that, when added to a compound or molecule, such as a protein or polypeptide, can increase the flexibility of the compound or molecule. A flexible linker can be a peptide or polypeptide. Other types of peptide linkers can be rigid linkers or cleavable linkers.

[0122] In some embodiments, a flexible peptide linker can include small polar (e.g., Ser, Thr) or non-polar (e.g., Gly) amino acids. A flexible peptide linker can have a sequence of Gly and Ser residues (e.g., a "GS" linker). An exemplary GS linker amino acid sequence is (Gly-Gly-Gly-Gly-Ser). n Other types of flexible linkers include KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, (Gly)8, GSAGSAAGSGEF, and (GGGGS)4 (Chen, Xiaoying, Jennica L. Zaro, and Wei-Chiang Shen. "Fusion protein linkers: property, design and functionality." Advanced drug delivery reviews 65.10 (2013):1357-1369).

[0123] In some embodiments, flexible linkers can include 2-aminoethoxyacetic acid (AEA), 5-aminovaleric acid (Ava), 8-amino-3,6-dioxaoctanoic acid (PEG2 or AEEA), 12-amino-4,7,10-trioxadodecanoic acid (PEG3), and the like.

[0124] In some embodiments, 6-aminohexanoic acid (Ahx) can be used as a flexible peptide linker (Markowska, Agnieszka, Adam Roman Markowski, and Iwona Jarocka-Karpowicz. "The Importance of 6-Aminohexanoic Acid as a Hydrophobic, Flexible Structural Element." International Journal of Molecular Sciences 22.22(2021):12122.).

[0125] In general, any of these flexible linkers can be used in the compounds described herein.

[0126] Conjugates The moieties described herein can be attached to active agents using a variety of structures. These structures may be referred to herein as "conjugates." Attaching an active agent to a moiety may be referred to as "conjugating" or "conjugation." In some instances, when biomolecules are involved, the conjugate structure may be referred to as a bioconjugate.

[0127] In some embodiments, the conjugate can be a chemical. In some embodiments, the conjugate can be non-cleavable or cleavable. The conjugate can be designed to release the active agent under certain stimuli, including environmental stimuli such as pH, redox conditions, etc., in the presence of a given enzyme.

[0128] In some embodiments, conjugation or bioconjugation is carried out using "click" chemistry (Hein, Christopher D., Xin-Ming Liu, and Dong Wang. "Click chemistry, a powerful tool for pharmaceutical sciences." Pharmaceutical research 25.10 (2008):2216-2230.).

[0129] In some embodiments, MFCO-N-hydroxysuccinimide esters can be used to attach active agents that are proteins (eg, antibodies) to moieties disclosed herein.

[0130] In some embodiments, the conjugate may include a sulfo-NHS ester, a biotin-NHS-ester, and the like.

[0131] Pharmaceutical Compositions The antibodies of the present invention that specifically bind to OPN protein or a fragment thereof can be administered in the form of a pharmaceutical composition to treat neurological disorders or neurodegenerative diseases. Principles and considerations involved in preparing therapeutic pharmaceutical compositions containing antibodies, as well as guidance in the selection of ingredients, are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R. Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0132] The specific dosage and treatment regimen for a given patient will depend on a variety of factors, such as the given antibody, variant or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, drug combinations, and the severity of the given disease being treated. The judgment of such factors by a medical practitioner is within the skill of the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmaceutical and pharmacokinetic principles well known in the art.

[0133] A therapeutically effective amount of an antibody of the invention can be the amount necessary to achieve a therapeutic goal. As mentioned above, this can be a binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody for its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The dosage of the antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is about 0.1 mg / kg to 100 mg / kg patient body weight, 0.1 mg / kg to 20 mg / kg patient body weight, or 1 mg / kg to 10 mg / kg patient body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to the foreign polypeptide. Thus, lower dosages and less frequent administration of human antibodies can be used. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue (e.g., brain) penetration by modifications such as, for example, lipidation. A typical range for therapeutically effective administration of an antibody or antibody fragment of the invention can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequency can range, for example, from twice daily to once a week.

[0134] When an antibody fragment is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is useful. For example, a peptide molecule that retains the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al, Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation can also contain two or more active compounds necessary for a given indication to be treated, for example, those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can contain an agent that enhances its function, such as a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.

[0135] The active ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively.

[0136] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0137] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time.

[0138] The antibody or agent of the present invention (also referred to herein as "active compound"), as well as derivatives, fragments, analogs, and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions typically include the antibody or agent and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0139] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, e.g., water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0140] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In embodiments, the composition is sterile and fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, can be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0141] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or combination of the ingredients listed above as necessary, followed by filtration sterilization.For example, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion solvent and other necessary ingredients listed above.For the preparation of sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces powder of active ingredient and any additional ingredients from the solution that has been previously sterile filtered.

[0142] Oral compositions include inert diluents or edible carriers. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterote; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring ingredient such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0143] For administration by inhalation, the compounds are 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.

[0144] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0145] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0146] In one embodiment, the active compound is prepared in a carrier that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are clear to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with 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. Pat. No. 4,522,811.

[0147] For the convenience of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dosage unit form.The dosage unit form used herein refers to a physically separate unit suitable as a unitary dose for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of the dosage unit form of the present invention is determined and directly depends on the specific characteristics of the active compound and the given therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for the treatment of an individual.

[0148] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0149] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0150] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0151] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and are for illustrative purposes only, since alternative methods can be used to obtain similar results.

[0152] Example 1 Example 1 - Definition of mouse microglial subsets that regulate neuronal development and pro-inflammatory responses in the brain summary Expression of Itgax (encoding the CD11c surface protein) and Spp1 (encoding OPN) has been associated with activated microglia in the healthy brain and in some neuroinflammatory disorders that can develop. However, it is unclear whether CD11c and OPN gene expression is the result of microglial activation or represents part of a genetic program expressed by a stable microglial subset. Here, we show that OPN production in the brain is driven by CD11c expression during perinatal life after incorporation of apoptotic neurons. - Small CD11c Differentiating from Progenitors + Our analysis shows that co-expression of OPN and CD11c genes represents a microglial subset that is expressed at birth and persists into late adulthood, independent of environmental activation stimuli. + Analysis of the contribution of OPN to the intrinsic functions of microglial subsets indicates that OPN is required for subset stability and execution of phagocytic and proinflammatory responses, in part through OPN-dependent engagement of the αVβ3 integrin receptor. OPN-producing CD11c as a functional microglial subset. + Defining microglia provides new insights into microglial differentiation in health and disease.

[0153] Statement of Importance CD11c with increased osteopontin gene expression + Microglia appear at different stages of brain development, aging and in some neurodegenerative disorders. It remains unclear whether the co-expression of CD11c and OPN results from microglial activation or represents part of a subset-specific genetic program. + We found that a microglial subset is formed prenatally upon the ingestion of apoptotic neurons. Our analysis also shows that it is a stable subset that requires OPN to mediate phagocytosis of synaptic proteins, proliferate, and express a proinflammatory phenotype. OPN-producing CD11c as a specialized microglial subset + Defining microglia provides new insights into the contribution of microglial differentiation to brain development and function in health and disease.

[0154] Introduction CD11c, also called integrin alpha X (encoded by Itgax), is a defining marker for dendritic cells (DCs). When paired with Itgb2, the heterodimeric receptor binds complement iC3b and mediates phagocytosis (1). A subpopulation of CNS-resident microglia that also expresses CD11c develops early in life and is a hallmark of microglial development in the healthy brain and in mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) (2-4). CD11c + Genes expressed by microglia include the Spp1 gene, which encodes osteopontin (OPN), a cytokine-like phosphoprotein that is a hallmark of both defensive and pathogenic immune responses in peripheral lymphoid tissues ( 4 – 8 ).

[0155] OPN is expressed as secreted (OPN-s) or intracellular (OPN-i) isoforms derived from a single OPN mRNA precursor (9) following activation of immune cells. Regulation of immune responses by OPN includes promoting proinflammatory responses following ligation of its canonical receptor, αvβ3 integrin expressed on macrophages (10-12). Production of OPN by DCs also regulates differentiation of T helper (TH) cell subsets (8), including TH17 cells, which contribute to the development of murine experimental autoimmune encephalomyelitis (EAE) (13). More recently, microglial production of OPN has been implicated in diverse CNS pathological disorders, including multiple sclerosis (3), spinal cord injury (14), and neurodegenerative disorders including AD and amyotrophic lateral sclerosis (ALS) (4, 15). CD11c + Although microglia are the major source of OPN production by activated microglia, it is unclear whether co-expression of CD11c and OPN is a hallmark of microglial activation or characterizes a subset-specific genetic program. This is a central question in understanding microglial development, as it involves either a selection of the genetic mechanisms regulating subset-specific differentiation rather than a marker of a transiently activated phenotype.

[0156] Here, the inventors have + OPN-producing microglia express CD11c after phagocytosis of apoptotic neurons (AN) during the perinatal period. - OPN - We report that CD11c is a small (<5%) subset that differentiates from precursors and is the only microglia-producing cell type in OPN throughout life. + OPN + The subset displays a stable phenotype at steady state and expresses a core gene program that is independent of microglial activation. + Analysis of the contribution of OPN to microglial function indicates that OPN regulates microglial proliferation and the development of a proinflammatory phenotype. OPN-produced CD11c as a functional subset rather than a transiently activated phenotype. +The definition of microglia represents a new approach to the analysis of microglial development and provides insight into the contribution of microglia to normal and dysregulated brain development.

[0157] result CD11c + Microglia form early in mouse brain development during phagocytosis of apoptotic neurons, independent of microglial activation In dendritic cells, the CD11c protein is an essential part of the iC3b heterodimeric receptor that mediates phagocytosis (1). CD11c-expressing microglia are detectable at birth but dramatically decrease by 3 months of age (3). Thus, we investigated the expression of CD11c in healthy C57BL / 6 mice from prenatal life to adulthood. + To track the development of microglia, we first verified microglial CD11c expression by flow cytometry. + CD45 low ) and macrophages (CD11b + CD45 high To distinguish between CD11b and CD21b-specific markers, we used the CCR2 marker, which is expressed by blood-derived macrophages but not by microglia (3, 16) and the microglia-specific marker Tmem119 (17). + CD45 high A subset was identified as macrophages, all expressing Tmem119 but not CCR2, CD11b + CD45 low Cells were identified as microglia. Fluorescence minus one (FMO) negative control and brain CD45 that do not express CD11c - Cells, including CD11b + CD45 low We confirmed the specificity of CD11c staining in the microglial population (Figure 1A). +We note that microglia arise late during embryogenesis (E18.5), increase to approximately 7-8% of total microglia by postnatal day 5 (P5), and then regress to nearly undetectable levels (<1%) during young adulthood. However, CD11c + Microglia re-emerge in older (6-9 months old) mice, representing approximately 10% of total microglia (Figure 1B).

[0158] Because uptake of apoptotic neurons (AN) can induce changes in the microglial gene program, including upregulation of Itgax RNA (encoding CD11c) (18), we hypothesized that CD11c - We investigated whether upregulation of CD11c expression by progenitors is a direct consequence of AN phagocytosis during early development. We identified CD11c expression by negative isolation using anti-CD11c-coated magnetic beads. - Microglia enriched for CD11c - Microglia were obtained (purity >99%) and incubated with fluorescently labeled AN for 72 h, after which CD11c was expressed. - The percentage of microglia was determined. In FIG. 1C, we determined the percentage of total CD11c - 23% of microglia are AN (AN + ), of which AN + 77.4% of microglia are CD11c + In contrast, CD11c cells that did not receive AN - Of the 77% of microglia, less than 1% expressed CD11c (upper panel, Fig. 1C). These findings also support the finding that total CD11c - Approximately 18% of microglia were CD11c after incubation with AN. + While microglia incubated for 72 h in the absence of AN continued to express CD11c + (Lower panel, FIG. 1C).

[0159] The upregulation of CD11c expression was not simply a consequence of microglial activation following, for example, phagocytosis or inflammation. Purified CD11c from P5 mice - Microglia were treated with various activating stimuli that mimic CNS inflammatory insults, such as LPS and amyloid β peptide (Aβ). These stimuli induced marked microglial activation, as judged by increased expression of both CD86 and MHCII (19), but not activated CD11c. - Microglia did not upregulate CD11c expression (Figure 1D, E). Collectively, these results suggest that CD11c expression is downregulated after AN intake at birth. + This indicates that microglial formation is independent of conventional microglial activation.

[0160] Next, we investigated the CD11c + We investigated interactions that may promote microglial formation. Phagocytic receptors expressed by developing microglia that may mediate the uptake and clearance of apoptotic cells include TAM (Tyro3, ​​Axl, MerTK) and integrin αVβ3 receptors (20, 21). We investigated the effect of specific inhibition of αVβ3 and TAM receptors on AN uptake, and the effect of CD11c - CD11c by microglia + The pan-TAM receptor αVβ3 inhibitor Cilengitide (Cil) or LDC1267 (LDC) inhibitors inhibited AN-uptake CD11c phenotype. - The combination of these two inhibitors reduced microglia by approximately 50%. - It further reduced microglial AN uptake to background levels (Fig. 1F, left panel). Consistent with the findings in Fig. 1C, we found that stimulation with AN reduced early CD11c - We noted that AN induced CD11c expression in approximately 20% of microglia, whereas in the absence of AN stimulation, CD11c expression was undetectable during the same period. Finally, inhibition of AN uptake by cilengitide or LDC1267 reduced CD11c expression. - CD11c by precursors+ The inclusion of both inhibitors reduced the acquisition of the phenotype by approximately 50–75% in each case, and the inclusion of both inhibitors completely prevented this phenotypic transition (Figure 1F, right panel). These findings suggest that phagocytosis of apoptotic cells is mediated by CD11c following interaction of AN with microglial receptors including αVβ3 and TAM. - →CD11c + This indicates that it can induce migration.

[0161] The stability of CD11c expression by microglia is regulated by OPN We verified microglial OPN expression by flow cytometry analysis. We used several controls, including an isotype control and, importantly, OPN-KO microglia as negative control cells and microglia selectively expressing the intracellular isoform of OPN (OPN-i-KI) as a positive control. WT and OPN-i-KI microglia show similar levels of OPN staining, whereas staining of microglia from OPN-KO donors or stained with the isotype control does not yield a detectable signal (Figure 2). We found that microglial OPN production is related to CD11c expression. + Restricted to microglia, CD11c - We noted that CD11c was not significantly expressed by microglia (Figure 3A). - From CD11c + AN-induced differentiation into microglia was also accompanied by a rapid upregulation of OPN production ( Fig. 3B ).

[0162] CD11c + To define the effect of OPN on phenotype, we upregulated CD11c IL-16 expression in OPN-KO and WT mice during early development and aging. + Microglia were characterized. OPN deficiency reduced CD11c expression in P5, 6-month-old, and 9-month-old WT mice by flow cytometry analysis. + This resulted in a 50% reduction in the proportion of microglia (Figure 4A). +and CD11c - In situ analysis of microglia revealed that CD11c + A reduction in microglial properties (50-75% reduction) was confirmed. CD11c specific staining was verified using a negative control (Figure 4B).

[0163] We further defined the contribution of OPN to the stability of CD11c expression by microglia isolated from P5 and 9-month-old WT and OPN-KO mice during a 7-day in vitro culture period. + The number of microglia was similar between WT and OPN-KO mice at day 0, P5, and 9 months of age (Figure 5A). + Microglia showed a 75% reduction in CD11c expression (Fig. 3C), whereas OPN-deficient CD11c from 9-month-old WT mice + Microglia showed a 50% reduction in CD11c expression (Figure 3D). Using organoid hippocampal slice cultures (OHSCs), we investigated the expression of microglial CD11c in a more physiological context. + The stability of the phenotype was examined. This system allows replacement of endogenous microglia after depletion of resident microglia from organotypic hippocampal tissue slices. We used CD11c phenotypes derived from WT or OPN-KO P5 and 9-month-old donors. + Microglia (>95% CD11c + ) to microglia. WT and OPN-KO mice expressed similar numbers of CD11c + Microglia were shown (Figure 5B). WT CD11c + Microglia express their CD11c + Retained phenotype but lacked OPN CD11c + The majority of microglia were not retained (Figures 3E and 3F).

[0164] We then demonstrated that CD11c signaling is a pathogenic microglial stimulus for Aβ, a hallmark of age-related neurodegenerative diseases. +Microglial responses were evaluated. We used CD11c + OPN-deficient CD11c microglia lose 60-75% of their CD11c expression + In contrast to microglia, we found stable expression of CD11c after in vitro Aβ stimulation and in OHSCs (Figure 3G, H). Collectively, these data indicate that stable expression of microglial CD11c may require co-expression of OPN.

[0165] CD11c + Genetic profiling of microglia We then used CD11c + We investigated whether microglia represent a distinct subset expressing a distinctive genetic profile. To that end, we used a cold isolation protocol to FACS-sort CD11c + and CD11c - RNA-seq analysis of microglia was performed and subsequently validated using flow cytometry (Figure 6). For example, CD11c from neonatal (P5) WT mice was + The transcriptomes of CD11c- and CD11c-microglia overlapped substantially, sharing 10,385 genes. However, a small number of genes (12–15) were CD11c- and CD11c-specific. - (12 genes), and approximately 15 genes were expressed by CD11c + Similarly, CD11c from 9-month-old mice was specifically expressed by microglia. + and CD11c - Microglia shared 12,072 genes, 19 of which were CD11c + CD11c was expressed exclusively by microglia (Figure 7A). We found that CD11c was expressed by newborn (P5) and aged (9-month-old) mice (Figure 7B). + CD11c shared by microglia and derived from donors of any age -We identified four genes that were not even expressed by microglia (Figure 7C). These included CD11c, which is maintained from neonatal to advanced age in the absence of exogenous inflammatory or infectious stimuli, and CD21c, which is expressed by microglia but not by inflammatory cells. + These genes are selectively expressed by microglia. These genes include CD36, an inflammatory response regulator (22), and CD209a, a regulator of phagocytic activity (5, 23), and are expressed at both the RNA and protein levels (Figure 7D, E). - Since deliberate activation of microglia did not induce expression of these core proteins (CD209 and CD36), CD11c + The expression of these proteins by microglia was independent of activation stimuli (Figure 7F). Furthermore, these signature genes were upregulated by CD11c after phagocytosis of AN. - Precursor CD11c + were upregulated by the offspring, indicating that upregulation of these genes accompanies the formation of this subset during the perinatal period ( Fig. 7G ).

[0166] OPN is CD11c + Regulating intrinsic functions of microglia Microglia may contribute to the elimination of excess neuronal synapses during neonatal brain development (24). + OPN + We found that microglia were highly phagocytic towards synaptosomes, whereas those from 9-month-old brains were not (Figure 8). We investigated whether OPN contributes to this microglial function. OPN is expressed as secreted (OPN-s) or intracellular (OPN-i) isoforms derived from different OPN translation initiation sites (25). We utilized OPN mutant mice with different OPN isoforms to express CD11c + We have described the contribution of OPN isoforms to microglial function. Complete OPN deletion suppresses CD11c +This resulted in a 45% reduction in synaptosome phagocytosis by microglia. OPN-i was not involved in this process (Figure 8A). Similarly, ex vivo analysis showed that CD11c + OPN + We found that microglia exhibited stable phagocytosis of synaptic proteins, including synaptophysin and PSD95, whereas 9-month-old WT mice did not. Deletion of OPN-s and both OPN-i and OPN-s alone did not suppress this CD11c + This resulted in an approximately 50% decrease in microglial function (Figure 8B).

[0167] CD11c + When we examined the contribution of OPN to microglial proliferation, selective deletion of OPN-s reduced CD11c expression from both newborn and aged WT mice. + It was found that the interaction between OPN-s and its canonical receptor, CD11c, was sufficient to substantially reduce microglial proliferation (Figure 8C, D). + We investigated whether OPN receptors may contribute to microglial function. CD44 and αVβ3 OPN receptors (26, 27) are not expressed by microglia at any age, whereas αVβ3 expression increases gradually during aging (Figure 9). Small molecule-mediated blockade of αVβ3 with a cyclic RGD inhibitor (cilengitide) markedly reduced OPN-dependent Ki-67 expression (Figure 8E) and upregulated CD11c + We showed that OPN-dependent proliferation of microglia reflects ligation of the αVβ3 receptor. + The robust proinflammatory response of microglia was dependent on OPN-s expression (FIG. 8F) and reflected engagement of the αVβ3 integrin receptor (FIG. 8G).

[0168] This OPN-producing CD11c + The age-dependent pro-inflammatory phenotype of microglial subsets indicates that they may contribute to neuroinflammatory disorders. Therefore, we investigated the mechanism by which CD11c +We investigated whether expression of OPN-producing microglia signature genes could be sustained in the context of chronic inflammatory stimuli occurring in 5XFAD mice, which recapitulate the amyloid pathology of Alzheimer's disease. + Microglia express their CD11c in the presence of 5XFAD pathology - We found that the representative core genes CD36 and CD209a were expressed at the protein level compared to their counterparts (Figure 10A, B). Furthermore, OPN production was significantly higher than CD11c from age-matched OPN-KO.5XFAD mice in vitro and ex vivo. + In contrast to the significant decrease in microglial stability (approximately 40%–80% decrease), CD11c + The CD11c phenotype also contributed to microglial stability (Figure ​(Figure10C). 10C). + Although stably expressed by microglia, OPN deletion resulted in a 40%-80% reduction in OPN in vitro and in OHSCs, respectively (Figure 10D).

[0169] Consideration Microglial expression of the CD11c receptor and production of OPN have been linked to microglial activation during the development of several neuroinflammatory diseases (3, 4, 7) and in response to exogenous stimuli (14). This could be the result of microglial activation or it could be part of a genetic program of a microglial subset that develops at or before birth and persists into late adulthood. Our experiments support the latter view, i.e., CD11c + These results support the idea that microglia are a stable subset programmed to produce OPN rather than a transiently activated phenotype.

[0170] We demonstrated that CD11c expression at birth during phagocytosis in AN in the absence of external activating stimuli - Small (<5%) OPN-producing CD11c cells differentiate from + Identifying microglial subsets. Initially formed during the perinatal period, they express CD11c +Microglia regress to nearly undetectable levels during young adulthood, then reemerge in late adulthood to comprise 10-15% of all microglia. + Microglia and late adult CD11c + Both microglia express a gene signature that is independent of cell activation in healthy mice.

[0171] Single-cell transcriptomics have described microglial subsets enriched for Itgax (encoding CD11c) at different ages and during the development of neurodegenerative diseases, in accordance with the description by Owens' group (3) (4-7), and CD11c + We found that microglia numbers peaked early after birth (P3–5) and declined to marginal levels by young adulthood (2–3 months). + Our analysis of microglia revealed that CD11c + We found that microglia undergo a transient shrinkage followed by reemergence and expansion to substantial numbers during normal aging.

[0172] Changes in the microglial gene transcription program following phagocytosis of apoptotic cells include upregulation of Itgax gene expression (18, 28), but observations in unselected microglial populations suggest that this is due to upregulation of CD11c - De novo expression of CD11c by precursors or from smaller populations + Whether this reflects microglial proliferation remains to be determined. - Analysis of the response of precursors to AN was determined by neonatal CD11c - We showed that induction of CD11c protein expression following AN phagocytosis by precursors was accompanied by OPN expression, indicating that this phenotype is a direct consequence of AN-induced differentiation. This view is supported by the upregulation of CD11c by various nonspecific stimuli. - Since deliberate activation of precursors failed to induce expression of the CD11c phenotype, +This is supported by the finding that microglia formation is independent of nonspecific microglial activation. It may be related that microglia in neonatal brain regions containing large amounts of apoptotic cells express high levels of Itgax / CD11c and phagocyte-related genes (3, 6, 29, 30). Furthermore, injection of apoptotic neurons, but not live neurons, Escherichia coli, or zymosan particles into the brains of B6 mice, induces a microglial phenotype that includes upregulation of Itgax (CD11c) (18), indicating that microglial induction of CD11c is a tightly regulated response to cell apoptosis. Without wishing to be bound by theory, the conversion of adult microglia in later life may be efficiently promoted by other stimuli, such as amyloid beta alone or a synergistic mixture of apoptotic neurons and amyloid beta.

[0173] CD11c + Phenotypic expression was dependent on OPN production, as judged by in vitro analysis and after transfer into microglia-free hippocampal tissue. Furthermore, CD11c expression at birth and in late adulthood was also observed. + Analysis of microglial gene profiles revealed a persistent gene signature independent of conventional activation stimuli. + Selective expression of these signature genes by microglia is associated with CD11c - Although not mimicked by deliberate activation of microglia, CD11c + and CD11c - Further transcriptome profiling and single-cell transcriptome analysis of microglia are required to better describe the genetic makeup of this subset in healthy brain development and in response to chronic inflammatory disorders. A stable phenotype and expression of a core gene program that persists from birth to late adulthood independent of exogenous stimuli is consistent with the expression of CD11c. + We show that microglia may be a microglial lineage. To directly address this question, we used adipose mapping techniques to identify CD11c expression in different reporter mouse models. +More directed investigations tracing the genetic history of microglia are needed ( 31 , 32 ).

[0174] CD11c + The microglial genetic program includes genes related to phagocytosis and inflammation (22, 23), as well as CD11c + This indicates that microglia are specialized to carry out these microglial functions. + We note that microglia may contribute to the elimination of neuronal synapses by phagocytosing synaptic proteins during early development and mediate proinflammatory responses during aging, possibly allowing the elimination of defective or inactive synapses. + Phagocytosis of synaptic proteins by microglia is suppressed in the absence of OPN, but is suppressed by CD11c in adults. + Microglial proliferative and proinflammatory responses reflect OPN engagement of the αVβ3 integrin receptor, and thus CD11c + These functions of microglial subsets are regulated by OPN. Analysis of OPN mutant mice that specifically express OPN isoforms demonstrated that OPN-s, but not OPN-i, is responsible for these OPN-dependent functions. + The promotion of inflammatory responses by microglia is reminiscent of a subset of dendritic cells that express high levels of CD11c and execute OPN-dependent inflammatory responses (33). + Our findings that microglia express the protein-level signature genes and OPN-dependent phenotype seen in healthy mice allow us to define their contribution to disease development.

[0175] In summary, the present inventors demonstrated that CD11c is the sole source of OPN in the brain. + Microglia express CD11c after ingesting apoptotic neurons at birth - Without wishing to be bound by theory, this OPN-producing CD11c +The microglial population is a novel subset that follows its stable phenotype and expression of a signature set of genes at birth and in late adulthood that is independent of deliberate activation. + The pro-inflammatory properties of microglia indicate that these cells may contribute to the pathogenesis of inflammatory diseases such as AD, amyotrophic lateral sclerosis, and Parkinson's disease.

[0176] Materials and Methods Mice. Wild-type C57BL / 6 (B6) and B6.Cg-Tg (APPSwFlLon, PSEN1 * M146L * L286V)6799Vas / Mmjax(5XFAD) mice were obtained from Jackson Laboratory. flstop (OPN-KO) mice and Spp1 flstop Ella cre (OPN-i-KI) mice were generated in our laboratory as previously described (34). OPN-KO.5XFAD were transfected with Spp1 flstop Mice were bred by mating with 5XFAD mice. All mice were housed under pathogen-free conditions. All experiments were performed in accordance with federal law and institutional guidelines approved by the Dana-Farber Cancer Institute (DFCI) Animal Care and Use Committee.

[0177] Isolation of microglia Microglia isolation by MACS. Adult mice were anesthetized with isoflurane and perfused transcardially with ice-cold PBS, after which the brains were removed and minced with a scalpel. The tissue was subjected to enzymatic dissociation using collagenase (300 U / mL, Worthington) or papain (20 U / mL, Worthington) and DNase I (60 U / mL, Worthington), after which a 30% Percoll gradient was performed to remove myelin and pelleted cells were resuspended in MACS buffer (PBS pH 7.2, 2 mM EDTA and 0.5% BSA). Total microglia were obtained by magnetic isolation using CD11b microbeads (Miltenyi). CD11c + and CD11c -For isolation of microglia, single cell suspensions were incubated with CD11c microbeads (Miltenyi) and the cells magnetically bound to the column using MACS were extensively washed, after which the magnetic field was released to separate the CD11c + The unbound fraction was then labeled with CD11b microbeads (Miltenyi) and separated using MACS isolation to separate the column-bound CD11c - CD11b + Cells were eluted using MACS buffer according to the manufacturer's protocol. Microglia were isolated for all experiments using this standard method unless otherwise stated.

[0178] Microglia isolation and fluorescence-activated cell sorting for RNA-seq analysis. Single cell suspensions were prepared as described (6). Briefly, mice were anesthetized with isoflurane and transcardially perfused, after which the brains were quickly excised and minced using a scalpel on ice, followed by approximately 20 rounds of Dounce homogenization in ice-cold HBSS. All tools used were pre-cooled, and all isolation steps were performed on ice to minimize microglial activation. The cell suspension was transferred to a pre-cooled 50 mL tube, passed through a 70 μm cell strainer, then transferred to a pre-cooled 15 mL tube and spun down at 500 g for 5 min at 4 °C. Debris and myelin were then removed using a modified cold Percoll gradient, and the cell pellet was resuspended in 10 mL of diluted ice-cold 40% Percoll (Sigma) and then spun at 500 g for 30 min. This technique yielded a microglial pellet at the bottom of the 15 mL tube while Percoll and myelin were simultaneously removed by vacuum aspiration. The cell pellet was washed with 10 mL of ice-cold HBSS and spun again at 500g for 5 min at 4°C. All samples were then resuspended in ice-cold FACS buffer (0.5% BSA, 1 mM EDTA in 1x PBS) for staining. Ghost dye Violet 510 (1:1000, Tonbo Biosciences) was used to exclude dead cells. To avoid non-specific staining, Fc receptors were blocked using CD16 / CD32 antibody (1:100, BD Biosciences). The single cell suspension was then stained with anti-CD11b antibody (1:100), anti-CD45 antibody (1:100) and anti-CD11c antibody (1:100) (Biolegend) for 20 min on ice, after which the samples were washed with ice-cold FACS buffer and spun down at 500g for 5 min. The cell pellet was resuspended in 5 mL of ice-cold FACS buffer and then sorted on a BD FACS Aria II using a 70 μm nozzle in purity mode at approximately 10,000 events / sec. After sorting, each sample was spun down and the cell pellet was immediately stored at -80°C until further processing.

[0179] For P5 mice, CD11c + Microglia and CD11c- Three biological replicates of microglia were sequenced. Samples were pooled from 58 mice. Duplicate CD11c + Microglia and CD11c - Microglia were sequenced. Samples were pooled from 44 mice (22M+22F).

[0180] Apoptotic Neurons (AN): CD11c - Induction of microglial differentiation and labeling. Primary mouse neurons were prepared from B6 embryos at embryonic day 16.5–17.5. Brain hemispheres were isolated and meninges were removed, followed by tissue digestion with 0.25% trypsin in HBSS for 15 min at 37 °C, followed by subsequent titration to obtain single cell preparations. The cell suspension was filtered through a 70 μm cell strainer, and cells were centrifuged at 600 g for 5 min. Cell density was determined using a hemocytometer, and cells were seeded in Neurobasal medium supplemented with 1 × B27 and 500 μM GlutaMax (Invitrogen). Half of the medium was replaced every 3 days. To induce apoptosis, primary cultured WT neurons were treated overnight with 300 μM NMDA, then carefully detached from the flask by repeated washing with PBS, followed by centrifugation, and the pellet was processed for labeling. Neurons (1 × 10 7 ) were carefully resuspended in 1 mL of PBS and incubated with 100 μg of dissolved labeling dye (pHrodo iFL Green, Life Technologies) for 2 h in the dark at room temperature. To block and capture residual dye, cells were diluted with PBS, collected by centrifugation, resuspended in 1 mL of FBS, and washed twice with PBS. Total apoptotic cell numbers were determined using trypan blue staining.

[0181] Flow cytometry Microglia staining for flow cytometry analysis. Microglia were stained with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) to exclude dead cells, followed by Fc receptor blocking using CD16 / CD32 antibodies (1:100, BD Biosciences) to avoid non-specific staining. Appropriate microglial surface markers such as anti-CD11b (1:100 Biolegend), anti-CD45 (1:100 Biolegend), anti-CD11c (1:50 Biolegend), anti-CD36 (1:100, Biolegend), anti-CD209a (1:100, Biolegend), anti-αV integrin (1:50, Biolegend), anti-β3 integrin (1:50, Biolegend), anti-CD86 (1:100, Biolegend) and anti-MHC II (1:100, Biolegend) were used for staining, followed by fixation and permeabilization for subsequent intracellular staining with anti-OPN (1:10, R&D Systems), anti-TNF-α (1:50, Biolegend) and intranuclear staining with anti-Ki-67 (1:100, Biolegend).

[0182] Validation of microglial OPN expression by flow cytometry analysis. Microglial OPN expression was validated in 9-month-old WT mice using a conventional intracellular staining protocol. Microglia were fixed and permeabilized with intracellular fixation and permeabilization buffer (eBioscience) and then incubated with PE-conjugated anti-OPN Ab (1:10, Cat.NO.IC808, R&D Systems) for 30 min at 4°C. Isotype control (1:10, PE-conjugated goat IgG) and OPN-KO microglia were used as negative controls. Microglia selectively expressing the intracellular isoform of OPN (OPN-i-KI) were used as positive controls.

[0183] Validation of microglial CD11c expression by flow cytometry analysis. Brain single cell suspensions from 9-month-old WT mice were stained with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and anti-CD16 / CD32 antibodies (1:100, BD Biosciences), followed by staining with anti-CD11b (1:100, Biolegend), anti-CD45 (1:100, Biolegend), anti-CD11c (1:50, Biolegend), anti-TMEME 119 (1:200, ABCAM), and anti-CCR2 (1:100, Biolegend). + Cells were gated from single / live cells and subsequently identified as microglia with CD11b + CD45 low and CD11b as macrophages + CD45 hi To distinguish between microglia and macrophages, we used the CCR2 marker, which is expressed by blood-derived macrophages but not microglia (3, 16). To further distinguish between these two cell types, we also included the microglia-specific marker Tmem119 (17). CD11b, which expresses CCR2 but not Tmem119, was also included. + CD45 high The cells were identified as macrophages, but all expressed Tmem119 but not CCR2, CD11b + CD45 low The cells were identified as microglia. + CD45 low To confirm the specificity of CD11c staining in microglial populations, we included an FMO negative control. To further validate the specificity of this strategy, we also performed a FMO assay using brain CD45, which mainly contains non-immune cells that do not express CD11c (e.g., neurons, astrocytes, oligodendrocytes). - Cells were also included as a negative control.

[0184] Flow cytometric detection of microglial phagocytosis of synaptic proteins. After perfusion, mouse brains were harvested, sectioned, and subsequently myelin was removed by centrifugation on a 30% Percoll gradient. Brain pellets were stained sequentially with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and subsequently incubated with microglial surface markers: anti-CD11b, anti-CD45, and anti-CD11c for 30 min. Stained samples were then fixed and permeabilized using intracellular fixation and permeabilization buffer (eBioscience). Intracellular staining was performed for the presynaptic marker anti-synaptophysin (Invitrogen, 1:100) or the postsynaptic marker PSD-95 (Invitrogen, 1:100), followed by Alexa Fluor 488 donkey anti-mouse IgG (H + L) Stained with secondary antibody (Invitrogen, 1:300). Samples were acquired on a CytoFLEX (Beckman Coulter) flow cytometer and analyzed with FlowJo v10 (Tree star).

[0185] Organotypic hippocampal slice cultures. Organotypic hippocampal slice cultures (OHSCs) were prepared as described (35). Briefly, hippocampal slices were prepared from neonatal (P3-P5) C57BL / 6 mice at a thickness of 350 μm and then incubated at 35 °C in 5% CO2. Clodronate liposomes (FormuMax) were used to deplete microglia from freshly prepared slice cultures, and freshly prepared OHSCs were incubated with 0.5 mg / mL clodronate liposomes at 35 °C for 24 h. OHSCs were then rinsed with warm PBS, followed by a change of medium (50% MEM, 25% HBSS, 25% normal horse serum, 0.2 mM glutamine, 100 U / mL penicillin, 100 mg / mL streptomycin, and 4.5 mg / mL glucose). Microglia-depleted OHSCs were maintained for 7 days before experiments. CD11c +Microglia were acutely isolated from P5 or 9-month-old WT and OPN-KO mice or 9-month-old 5XFAD, OPN-KO.5XFAD mice. After isolation, microglia were carefully resuspended in culture medium to a final concentration of 2000 cells / μl. Each microglia-free OHSC was supplemented with 4000 cells. CD11c + Microglia-reconstituted OHSCs were incubated in the presence or absence of 2 μl of synthetic human Aβ peptide 1-42 (AnaSpec, 15 μM stock) × 7d. Aβ treatment was repeated four times, every other day, so that each slice was treated with a total of 8 μl of Aβ peptide.

[0186] Immunofluorescence staining. Transcardial perfusion with cold PBS was performed on P5 and 9-month-old WT and OPN-KO mice, followed by removal of the brains and fixing them in 4% paraformaldehyde (PFA) solution overnight at 4°C. The fixed brains were then rinsed with PBS and dehydrated in 30% sucrose overnight at 4°C. Brain tissue was embedded using OCT compound (Sakura Finetek) and serial sagittal cryosections (10 μm) were cut using a Cryostat (Leica). Brain cryosections were permeabilized with PBST buffer (PBS containing 0.3% Triton X-100) for 1 h. OHSC sections were fixed in 4% PFA solution for 30 min and permeabilized with PBST buffer for 3 h. After 1 h incubation in blocking solution containing 5% normal donkey serum (Jackson ImmunoResearch Lab) in PBST to prevent non-specific binding, cryosections or OHSC sections were incubated with the appropriate primary antibodies: rabbit anti-Iba-1 (1:1000, WAKO), biotin anti-mouse CD11c (N418, 1:50, Biolegend) for 24 h. To amplify the CD11c signal, sections were washed with PBS and incubated overnight with biotin anti-mouse CD11c, followed by 1 h incubation with HRP-conjugated streptavidin, and then incubated for 5 min with Alexa Fluor 488 tyramide to generate high density Alexa Fluor 488 labeling of CD11c protein in situ using the Tyramide SuperBoost Kit (Invitrogen). Sections were then incubated for 1 h with Alexa-594-conjugated donkey anti-rabbit IgG (1:500, Invitrogen). To exclude nonspecific immunofluorescence signals, immunofluorescence of brain slides incubated without anti-CD11c primary Ab or tyramide signal amplification (TSA) was included as a negative control. After using DAPI (Invitrogen) as a nuclear counterstain (10 min incubation), samples were analyzed using an Olympus fluorescence microscope.

[0187] RNA profiling RNA extraction. Total RNA was extracted from FACS-sorted cell pellets using the RNeasy Plus Universal Mini Kit according to the manufacturer's instructions (QIAGEN). Library preparation, sequencing reactions and bioinformatic analysis (gene hit counts) were performed at GENEWIZ, LLC. (South Plainfield, NJ, USA) as follows.

[0188] Library preparation. Extracted RNA samples were quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was confirmed using an Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). PolyA selection method: P5 WT CD11c RNA was purified using the NEBNext Ultra RNA Library Prep Kit for Illumina according to the manufacturer's instructions (NEB, Ipswich, MA, USA). + and CD11c - RNA sequencing libraries of microglial samples were prepared. Briefly, mRNA was first enriched with oligo(dT) beads, and then the enriched mRNA was fragmented at 94°C for 15 min. First- and second-strand cDNA were then synthesized, cDNA fragments were end-repaired and adenylated at the 3' end, and universal adapters were ligated to the cDNA fragments, followed by index addition and library enrichment by limited cycle PCR. Sequencing libraries were validated on an Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA) and quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).

[0189] Ultra-low input method: The number of cells obtained from adult mice is very limited. + and CD11c - Microglial samples were processed with the SMART-Seq v4 Ultra Low Input Kit (Clontech, Mountain View, CA) for full-length cDNA synthesis and amplification for sequencing and the Illumina Nextera XT library preparation for sequencing library preparation. Briefly, cDNA was fragmented using transposase and adapters were added, followed by limited cycle PCR to enrich and index the cDNA fragments. The final libraries were evaluated on an Agilent TapeStation.

[0190] HiSeq Sequencing. After the sequencing libraries were clustered on the flow cell lanes, the flow cell was loaded onto an Illumina HiSeq instrument (4000 or equivalent) according to the manufacturer's instructions. Samples were sequenced using a 2 x 150 bp Paired End (PE) configuration, and image analysis and base calling were performed by HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from the Illumina HiSeq were converted to fastq files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

[0191] RNA-Seq data analysis. Mapping and gene counting were performed by GeneWiz. After inspecting the quality of the raw data, sequence reads were trimmed to remove low-quality adapter sequences and nucleotides using Trimmomatic v.0.36. Trimmed reads were mapped to the Mus musculus reference genome (ENSEMBL) using STAR aligner v.2.5.2b, a splice aligner that detects and incorporates splice junctions to align the entire read sequence. BAM files were generated and unique gene hit counts were calculated using Counts (Subread package v.1.5.2). Only unique reads falling within exon regions were counted. Differential expression was considered significant at an FDR-adjusted p-value <0.05.

[0192] Quantitative PCR. CD11c was assayed using the RNeasy Plus Universal Mini Kit according to the manufacturer's instructions (QIAGEN). + and CD11c - Microglial RNA was extracted. cDNA was prepared by reverse transcription from 35ng RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Real-time quantitative PCR was performed using the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems) for selected core genes using 5μl cDNA, 4.92μl PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) and 0.08ul primers (IDT, working concentration: 200nM) per reaction. Normalized to β-actin ΔΔ Gene expression levels were compared using the Ct method.

[0193] In vitro synaptosome phagocytosis assay Synaptosome isolation and labeling. Synaptosomes were isolated from WT mice using Syn-PER Synaptic Protein Extraction Reagent (Thermo Scientific) according to the manufacturer's instructions. For pHrodo labeling, dissolved pHrodo iFL green (Life Technologies) was incubated with synaptosomes at room temperature for 1 h on a shaker in PBS protected from light at a ratio of 20 μg pHrodo per mg synaptosomes. After removing unconjugated pHrodo by washing with PBS, pHrodo-conjugated synaptosomes were resuspended in PBS containing 5% DMSO, aliquoted, and stored at -80°C until use.

[0194] Microglial phagocytosis of synaptosomes. Microglia isolated from P5 and 9-month-old WT, OPN-KO, and OPN-i-KI mice were cultured at 1 × 10 5 Cells were incubated with 136 μg of pHrodo Green-labeled synaptosomes per cell for 1 h, followed by staining with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and anti-CD11c (1:50, Biolegend) for 30 min. + Microglial phagocytosis was assessed by flow cytometry analysis.

[0195] Quantification and statistical analysis. Data in figures are presented as mean ± sem. Statistical analysis was performed using Prism (GraphPad Prism version 9.0). Quantification of fluorescent microscopy images was performed using ImageJ. Statistical analysis for comparison of mean values ​​of multiple groups was performed by one-way ANOVA or two-way ANOVA with Bonferroni's multiple comparison test using GraphPad Prism. Comparison between two groups was analyzed by two-tailed Student's t-test. P values ​​< 0.05 were considered statistically significant. All statistical details of the experiments, including the statistical tests used, exact values ​​of sample sizes can be found in the figure legends.

[0196] Data availability. All protocols and reagents are detailed in the manuscript and Supporting Information. RNA-seq data have been deposited in the NCBI Gene Expression Omnibus and are accessible via GEO Series accession number: GSE190713.

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I.M.Chiu et al.,A neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model.Cell Rep 4,385-401(2013). M.Mizutani et al.,The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood.J Immunol 188,29-36(2012). M.L.Bennett et al.,New tools for studying microglia in the mouse and human CNS.Proc Natl Acad Sci U S A 113,E1738-1746(2016). S.Krasemann et al.,The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases.Immunity 47,566-581 e569(2017). A.M.Jurga,M.Paleczna,K.Z.Kuter,Overview of General and Discriminating Markers of Differential Microglia Phenotypes.Front Cell Neurosci 14,198(2020). L.Fourgeaud et al.,TAM receptors regulate multiple features of microglial physiology.Nature 532,240-244(2016). S.Y.Park,I.S.Kim,Engulfment signals and the phagocytic machinery for apoptotic cell clearance.Exp Mol Med 49,e331(2017). E.Grajchen et al.,CD36-mediated uptake of myelin debris by macrophages and microglia reduces neuroinflammation.J Neuroinflammation 17,224(2020). D.Schulz,Y.Severin,V.R.T.Zanotelli,B.Bodenmiller,In-Depth Characterization of Monocyte-Derived Macrophages using a Mass Cytometry-Based Phagocytosis Assay.Sci Rep 9,1925(2019). E.Favuzzi et al.,GABA-receptive microglia selectively sculpt developing inhibitory circuits.Cell 184,5686(2021). M.Inoue,M.L.Shinohara,Intracellular osteopontin (iOPN)and immunity.Immunol Res 49,160-172(2011). G.F.Weber,S.Ashkar,M.J.Glimcher,H.Cantor,Receptor-ligand interaction between CD44 and osteopontin (Eta-1).Science 271,509-512(1996). E.Ruoslahti,M.D.Pierschbacher,New perspectives in cell adhesion:RGD and integrins.Science 238,491-497(1987). S.R.Anderson et al.,Developmental Apoptosis Promotes a Disease-Related Gene Signature and Independence from CSF1R Signaling in Retinal Microglia.Cell Rep 27,2002-2013 e2005(2019). L.D.White,S.Barone,Jr.,Qualitative and quantitative estimates of apoptosis from birth to senescence in the rat brain.Cell Death Differ 8,345-356(2001). N.Hagemeyer et al.,Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood.Acta Neuropathol 134,441-458(2017). P.B.Stranges et al.,Elimination of antigen-presenting cells and autoreactive T cells by Fas contributes to prevention of autoimmunity.Immunity 26,629-641(2007). L.Madisen et al.,A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci 13,133-140(2010). E.Kourepini et al., Osteopontin expression by CD103- dendritic cells drives intestinal inflammation.Proc Natl Acad Sci USA 111,E856-865(2014). JW Leavenworth, B. Verbinnen, J. Yin, H. Huang, H. Cantor, A p85alpha-osteopontin axis couples the receptor ICOS to sustained Bcl-6 expression by follicular helper and regulatory T cells. Nat Immunol 16,96-106(2015). S. Hellwig et al., Forebrain microglia from wild-type but not adult 5xFAD mice prevent amyloid-beta plaque formation in organotypic hippocampal slice cultures.Sci Rep 5,14624(2015).

[0198] Example 2 Example 2 - Osteopontin-producing microglia contribute to Alzheimer's disease overview Microglia are the resident immune cells in the brain, and dysregulated microglial activation is a cardinal feature of Alzheimer's disease (AD). 1 Despite abundant evidence for a key role in AD, the contribution of this cell type to disease pathology is poorly understood. Evidence that microglia may exert a protective effect comes from the observation that disease-associated microglia (DAMs) can phagocytose amyloid beta (Aβ) plaques. 2、3 In contrast, there is evidence that microglia are pathogenic, based on the observation that elimination of microglia prevents Aβ seeding and plaque formation in the 5XFAD mouse model. 4Our experiments indicate that microglial influences reflect separate contributions by protective and pathogenic microglial subsets that can be distinguished according to their developmental history and functional phenotype.

[0199] Although many disease-associated genes have been identified in mouse models of AD, most have not been confirmed in human AD experiments. The Spp1 gene (encoding OPN) is an exception, as it appears to be strongly upregulated by microglia in both animal models of AD and in human disease. 2、5~8 We recently characterized microglial subsets by expression of the surface marker CD11c, the sole producer of osteopontin (OPN), in the healthy mouse brain during early development and adulthood. 9 .

[0200] OPN is also expressed by peripheral dendritic cells and macrophages, where it can regulate inflammatory and autoimmune responses. 10~12 Steinman et al. implicate OPN in neuroinflammatory and neurodegenerative disorders such as multiple sclerosis. 13、14 However, the contribution of OPN-producing microglia to neurodegenerative diseases remains unclear.

[0201] Here, the present inventors have identified pathogenic CD11c that leads to AD pathology. + OPN + Defining microglial subsets: CD11c in 5XFAD mice +Genetic deletion of OPN production by microglia inhibits the production of inflammatory cytokines and promotes TREM2-dependent microglial uptake of amyloid fibrils and associated lysosomal activation. Targeting this OPN-dependent pathway leads to increased lysosomal degradation of Aβ fibrils and extrusion of compacted Aβ protein into the brain parenchyma, resulting in a reduction of diffuse Aβ plaques and a marked improvement in cognitive function. We extended these mouse findings to the human disease using clinically and neuropathologically characterized brain tissue from AD patients, mild cognitive impairment patients, and cognitively normal controls (Mt. Sinai Brain Repository). This analysis was supported by the inclusion of human CD11c + We showed that OPN production by microglia is closely correlated with the progression of both clinical dementia and AD neuropathology. Collectively, these findings suggest that CD11c expression may be important in reducing diffuse toxic plaques and inhibiting neuroinflammation. + These results suggest that targeting microglial production of OPN may be an effective treatment for AD.

[0202] result OPN contributes to AD pathology and cognitive impairment in 5XFAD mice To identify the cellular source of OPN during disease progression in the brains of 5XFAD mice, we measured OPN expression by astrocytes, neurons, and microglia at 3, 6, and 9 months of age. Astrocytes and neurons did not produce detectable amounts of OPN, but microglial production of OPN was stable and increased with disease progression (Figure 11a). There was a 10-20 fold increase in OPN mRNA and a 2-3 fold increase in OPN protein by 9 month old 5XFAD microglia compared to age-matched B6-WT controls (Figure 11b, c), indicating that microglial OPN production closely parallels disease onset.

[0203] CD11c-coexpressing microglia are the sole producers of OPN in the healthy mouse brain 9 The present inventors found that microglial OPN production in 5XFAD mice was also related to this CD11c+ was restricted to the microglial subset, which was found to increase dramatically during the first 6 months of disease progression compared to relatively low levels in healthy age-matched controls (FIGS. 11d, 11e).

[0204] We used 5XFAD transgenic mice with Spp1 flstop (OPN-KO) mice 15 We generated OPN-KO.5XFAD mice after crossing with and confirmed OPN deletion at both the gene and protein levels (Fig. 12). Genetic deletion of OPN in 5XFAD mice reduced microglial production of TNF-α to levels similar to age-matched healthy (B6-WT) controls (Fig. 11f). Examination of Aβ plaque area in OPN-KO 5XFAD mice revealed a 5- to 3-fold reduction in both cortex and hippocampus compared to age-matched 5XFAD controls at 6 and 9 months of age (Fig. 11g, h), indicating that OPN can inhibit Aβ clearance in 5XFAD mice. Anti-Aβ mAb 6E10 (to identify both diffuse and condensed forms of Aβ plaques) and Thioflavin-S (to identify β-sheet + Aβ plaques 3 Analysis of brain cryosections co-stained with OPN (which identifies only OPN-positive mice) also revealed that genetic deletion of OPN substantially reduced the rate of diffuse plaque development at 9 months of disease (Figure ​(Figure11i).11).

[0205] Neuritic dystrophies are caused by the amyloid precursor protein (APP). 16 A central component of 5XFAD disease pathology consists of swollen, bulbous neurites composed of dysfunctional axons and terminals expressing NF-κB, which correlate with the clinical severity of dementia. 17、18We noted that the number of dystrophic neurites per plaque was reduced by about 50% in OPN-KO.5XFAD mice (Fig. 11j, 11k). Because an increase in the number of dystrophic neurites closely correlates with cognitive decline, we measured the effect of OPN expression on cognitive function in 9-month-old 5XFAD mice using a water T-maze to assess spatial learning and memory (acquisition trials) and cognitive flexibility (reversal trials). 19、20 OPN deletion markedly improved these cognitive functions, as indicated by a 40-50% increase in correct choices made by OPN-KO.5XFAD mice in both the acquisition and reversal phases of these trials (Figure S11l). Collectively, these data indicate that OPN deletion substantially reduces microglial proinflammatory responses, diffuse Aβ plaques and dystrophic neurites, and improves cognitive function in 5XFAD mice.

[0206] Pathogenic CD11c in 5XFAD brains + OPN production by microglia promotes proinflammatory responses and inhibits Aβ uptake CD11c + Since microglia are the sole cellular producers of OPN in the mouse brain (Fig. 1d, Fig. 11a-c), the pathogenic effects of OPN may be due to the CD11c + It may depend on the development and function of microglial subsets.

[0207] The present inventors purified CD11c from 9-month-old 5XFAD and OPN-KO.5XFAD mice. + CD11c in 5XFAD disease according to RNA-Seq analysis of microglia + To define the contribution of OPN to the microglial phenotype, we identified CD11c +FACS gating of microglia was validated (Figure 13). OPN deficiency resulted in downregulation of inflammatory response-related genes (e.g., Tnfrsf9, il1b, Ccl1) and upregulation of phagocytosis-related genes such as Trem2, MerTk, CD68 and Ctsb (Figure 14a and Table 1), which contain core components of molecular pathways involved in Aβ uptake and lysosomal compaction. 3 Table 1 shows the CD11c expression levels in 9-month-old OPN-KO.5XFAD mice compared to FXFAD mice. + Contains a list of differentially expressed genes (DEGs) in microglia. 2,985 DEGs were identified. Gene expression was considered upregulated if log2FC was >1 and downregulated if log2FC was -1. DEGs were considered significant with an FDR adjusted p-value <0.05.

[0208] Flow cytometry analysis showed that the majority (80%) of microglia in brains from 9-month-old 5XFAD mice expressed neither CD11c nor OPN (double negative, DN), whereas approximately 15–20% expressed both (double positive, DP) and less than 5% expressed CD11c. + OPN - The present inventors showed that a significant number of CD11c - OPN + We then determined the proportion of each microglial subset in the cortex and hippocampus (C / H), the brain regions most affected by 5XFAD disease, and furthermore, using brain cryosections from 9-month-old 5XFAD mice, the percentage of microglial subsets in the periplaque region (25 mm from the Aβ plaque core). 21 The proportion of each subset present within the microglial CD11c-specific immunofluorescence signal was evaluated using several negative controls, followed by CD11c + OPN + , CD11c + OPN - and CD11c - OPN - Microglial subsets were stained and assessed (FIG. 15a, b).

[0209] CD11c + OPN + Approximately 60% of the (DP) microglia subset was present in the C / H region, and virtually all of these DP microglia were located within the periplaque region. Analysis of Aβ uptake by these periplaque DP microglia revealed that only approximately 12% contained ingested Aβ (Figure S14c, d). + Subset-CD11c + OPN - A significant proportion (approximately 70%) of CD11c- cells were also present within the C / H region, with the majority localized within the periplaque region. + OPN - Over 90% of microglia contained ingested Aβ material. + In contrast to the microglial subset, CD11c - OPN - The majority of the (DN) subset (>70%) was located in brain regions outside the C / H region. Analysis of the DN subset within the C / H region also showed that approximately half were located near or within periplaque regions, but only approximately 2% contained Aβ material (Fig. S4c, d).

[0210] Both CD11c + As microglial subsets are closely associated with Aβ plaques (Fig. 14d), we characterized their phenotype in more detail by flow cytometry. + OPN + (DP) expressed low levels of TNF-α but not TREM2 + CD11c significantly increased the proportion of microglia + OPN - These subsets showed substantial TNF-α production and reduced TREM2 expression compared to the CD11c subset (Figures 14e, 14f and 16a). - OPN - (DN) microglia comprise approximately 80% of total brain microglia, but do not produce detectable TNF-α and express marginal levels of TREM2 (Figure 16b). +Direct comparison of TREM2 and TNF-α expression by microglia revealed pro-inflammatory (i.e., TNF-α + )CD11c + Microglia have about 5% TREM2 + Cells containing TNF-α - Approximately 15% of microglia express TREM2 + These analyses revealed that DP CD11c + OPN + A pro-inflammatory phenotype of microglia was associated with reduced Aβ uptake (Fig. S14c, d), whereas CD11c cells lacked significant pro-inflammatory components. + OPN - Microglia are shown to exhibit stable levels of Aβ uptake.

[0211] Because genetic deletion of OPN reduces microglial production of TNF-α to levels seen in non-diseased mice (Fig. 1f), we investigated the relationship between OPN and the pro-inflammatory TNF-α phenotype. OPN-dependent ligation of macrophage αVβ3 integrin receptor promotes a pro-inflammatory phenotype and reduced uptake of apoptotic debris. 22、23 OPN 9、24 Microglial expression of the canonical αVβ3 integrin receptor for OPN was substantially increased in 5XFAD mice, whereas other OPN receptors, CD44 and αVβ5, were barely detectable (Fig. 16c). Addition of rmOPN increased the expression of CD11c + It induced TNF-α expression by microglia, which was abolished by treatment with cyclic RGD blockade of αVβ3 integrin (Cilengitide) (FIG. 16d). - OPN - Microglia expressed negligible amounts of αVβ3 but not CD11c, consistent with the virtual absence of proinflammatory TNF-α expression by this subset. + Subset (CD11c + OPN - and CD11c + OPN +) expressed higher levels (>30%) of αVβ3 (Fig. 14h). + To directly define the contribution of OPN to the microglial phenotype, we examined microglia from OPN-KO.5XFAD mice. We investigated CD11c + Microglia upregulate Aβ along with a 50% reduction in TNF-α production compared to 5XFAD mice (Figure ​(Figure14j). + It was noted that the mice showed a substantially increased uptake of the substance (Figure 14i).

[0212] In summary, (a) OPN deficiency reduces the expression of αVβ3 + CD11c + TNF-α in microglia + resulting in a decrease in the proportion of cells expressing TREM2 + (b) CD11c that lacks the OPN receptor, resulting in an increased proportion of microglia + Microglia do not produce TNF-α (Fig. 14k, 14l and 16e). These findings suggest that OPN mediates the expression of CD11c + They show that engagement of its (αVβ3) receptor on microglia can promote TNF-α production and reduce TREM2 expression.

[0213] Taken together, these findings reveal distinct contributions of three major microglial subsets to 5XFAD disease. - OPN - Although the microglial subset represents the major subset in the brain (approximately 80%), it does not take up Aβ, does not produce TNF-α, and is not a major component of Aβ pathology. In contrast, CD11c + (OPN + and OPN - Although microglia account for a relatively small proportion (approximately 20%) of microglia in the total brain, more than 60% of these microglia are located within plaque regions. +OPN production by microglial subsets further refines their distinct functional phenotypes within and around plaque regions. + OPN + Although microglia produce high levels of TNF-α, relatively few contain ingested Aβ or express TREM2, consistent with their pathogenic contribution to disease. + OPN - Although microglia do not produce significant levels of TNF-α, they actively phagocytose Aβ and may exert disease-preventive rather than disease-promoting effects (summarized in Figure 17).

[0214] OPN inhibits Aβ plaque compaction via suppression of the TREM2-lysosomal phagocytosis pathway Recent evidence indicates that the TREM2 pathway enhances lysosomal degradation of amyloid fibrils, leading to increased extrusion of condensed Aβ at the expense of diffuse morphology of Aβ plaques in 5XFAD mice. 25、26 We investigated whether the reduction in diffuse plaques previously observed in OPN-KO.5XFAD brains (Fig. 11i) reflected increased activation of this TREM2-lysosomal pathway. Analysis of TREM2 expression in situ on brain cryosections confirmed the flow cytometry analysis: CD11c from OPN-KO.5XFAD + Microglia expressed 2-3 times more TREM2 compared to 5XFAD mice (Fig. 19a,b). This increase was associated with enhanced lysosomal activation as judged by increased expression of the CD68 lysosomal activating glycoprotein (Fig. 19c,d) and a 3-4 fold increase in lysosomal expression of cathepsin B, a lysosomal cysteine ​​protease that cleaves and degrades Aβ (Fig. 19e,f) in microglia from OPN-KO.5XFAD mice compared to age-matched 5XFAD mice. 27、28 Taken together, these data suggest that OPN-dependent downregulation of the TREM2-phagolysosomal Aβ compaction pathway and genetic deletion of OPN reduce total plaque (6E10 +) Diffuse plaques in (6E10 + Thio-S - ) which may explain our findings (e.g., FIG. 11i).

[0215] We then demonstrated that the interaction between recombinant OPN and CD11c + We investigated whether TREM2 expression by microglia could be inhibited. We found that TREM2 expression was rapidly inhibited by rmOPN, and this decrease was reversed by the addition of anti-OPN Ab (Fig. 18a,b). Furthermore, rmOPN also inhibited lysosomal activation, as judged by the reduced expression of CD68 (Fig. 18c). The suppression of TREM2-lysosomal activation (Fig. 19a-d and Fig. 18a-c) was completely reversed by anti-OPN Ab, whereas CD11c expression was completely reversed by anti-OPN Ab. + Substantial reduction in Aβ degradation in microglial lysosomes [(Aβ MFI 1h -Aβ MFI 24h ) / Aβ MFI 1h ] (Figure 18d).

[0216] To directly test the contribution of OPN to microglial plaque compaction, we used thioflavin-S to distinguish compacted plaques from total plaques and to characterize Aβ in a β-sheet conformation. + The inventors identified the aggregates. + Thio-S + Area / 6E10 + Judging from the significant increase in compact plaque area (Fig. S19i), we found that the 40-45% reduction in total plaque area in OPN-KO.5XFAD mice shown in Figures 3g and 3h was accompanied by an increase in compact plaque area (Fig. S19i) (Fig. S19j).

[0217] Taken together, these data suggest that OPN-mediated inhibition of TREM2-dependent lysosomal activation, which leads to impaired microglial compaction of Aβ plaques and increased diffuse plaque formation, may promote 5XFAD disease. + CD11c +This indicates that this is a pathogenic pathway promoted by microglia.

[0218] CD11c + Increased OPN production by microglia correlates with disease severity and neuropathology in brain tissue from AD patients To extend these findings from mouse models of AD to the human disease, we analyzed brain tissue from clinically and neuropathologically confirmed AD patients with a clinical dementia rating (CDR) ≥ 1 at death (Tables 2 and 3 characterization of the cohort), as well as mild cognitive impairment patients (MCI, CDR = 0.5) and cognitively normal controls (CDR = 0). Measurement of OPN in middle frontal gyrus homogenates revealed a three-fold increase in OPN expression in AD patient brains compared to that of cognitively normal control subjects. Increased OPN expression was also observed in AD patient brains compared to MCI patient brains, but did not reach statistical significance (Figure 20a). Correlation analysis of brain OPN expression and CDR scores showed that increased OPN expression was positively correlated with dementia severity (Figure 20b).

[0219] Next, the inventors 29 Immunofluorescence analysis of human brain sections from the middle frontal gyrus, an area that is early affected by + OPN + (DP) The percentage of microglia was determined. Normal controls and MCI subjects had similar percentages of CD11c + OPN + Microglia (CD11c + OPN + Iba-1 + ) in brain sections from AD patients compared to sections from normal controls. + OPN + We found that microglia increased three-fold (Fig. 20c, 20d). Furthermore, CD11c cells derived from AD patients were + OPN + The percentage of microglia was significantly higher than that from MCI patients (Fig. 20d), and CD11c + OPN +Our results indicate that the percentage of microglia is a more sensitive parameter for discriminating AD patients from those suffering from MCI than simple OPN levels (Figure 20c). + OPN + This is further supported by the very strong correlation between the percentage of microglia and the CDR score (Figure 20e).

[0220] We also investigated (a) OPN levels and (b) CD11c expression in the middle frontal gyrus with neuritic plaques. + OPN + Correlation was examined between the percentage of microglia and neurofibrillary tangle density assessment determined postmortem by a neuropathologist prior to depositing the samples in the Mount Sinai Brain Repository as described. 30 We found that higher brain OPN levels correlated with higher neuritic plaque levels (r=0.4919, p=0.0043) and neurofibrillary tangle scores (r=0.4884, p=0.0046) (Fig. 20f, h). Similar to the association seen with dementia severity, CD11c + OPN + (DP) There was a stronger correlation between the percentage of microglia and the density of neuritic plaques (r=0.8226, p<0.0001) and neurofibrillary tangles (r=0.7434, p<0.0001) (Fig. 20g, i). These findings were consistent with the CD11c + OPN + This further supports the view that the percentage of microglia may be a sensitive marker of both clinical severity and neuropathology of AD.

[0221] Consideration Microglial dysregulation, characterized by persistent inflammatory responses and impaired Aβ plaque processing, may contribute to cognitive decline in the setting of neuritic dystrophies and AD 1、31 We previously noted that a small microglial subset expressing CD11c, originally defined by Owens' group, is the sole producer of OPN in brain tissue from healthy mice.9 Here, we show that OPN production similarly upregulates CD11c in the 5XFAD model of AD during disease development. + Here, we report that OPN production is similarly restricted to microglia, upregulating CD11c in the 5XFAD model of AD during disease development. + We report that CD11c is restricted to microglia. + OPN + OPN production by a subset may reflect an enhanced proinflammatory response in activated lysosomes and impaired TREM2-dependent Aβ plaque anchoring. These findings provide new insights into the molecular mechanisms that drive AD-associated cognitive impairment and, without being bound by theory, highlight OPN as a tractable therapeutic target in AD.

[0222] Based on CD11c expression and OPN production, microglia are divided into three major subsets: CD11c + OPN + , CD11c + OPN - and CD11c - OPN - Can divide into microglia. CD11c - OPN - Although the CD11c subset represents over 80% of microglia in the entire brain, less than 30% reside in the cortex / hippocampus of the 5XFAD mouse brain, with approximately 10% located within the periplaque region. - OPN - Fewer than 2% of microglia take up Aβ and do not produce appreciable TNF-α, so this CD11c - OPN - Microglia are a homeostatic subset that contribute to 5XFAD pathology. In contrast, expression of OPN downregulates CD11c + Microglial ablation identifies pathogenic and protective subsets. CD11c + OPN -Although microglia constitute a minor population (~3%) in the whole brain, ~70% of this subset resides in the cortex / hippocampus, with the majority of these cells (~65%) located in the periplaque region. These CD11c + OPN - Microglia may be protective, as they stably take up Aβ (~60%), express high levels of TREM2, and produce negligible levels of TNF-α. + OPN + The DP microglia subset is also significantly enriched in the periplaque region (approximately 60%), but while only approximately 5–7% take up Aβ, nearly 60% produce TNF-α, express low levels of TREM2, and upregulate CD11c. + OPN + Supporting the view that microglia are a pathogenic microglial subset.

[0223] TREM2 is a novel inhibitor of Aβ 32、33 Since CD11c can promote microglial uptake of + OPN - Strong expression of TREM2 by microglia may contribute to stable Aβ uptake by these cells. In contrast, DP CD11c + OPN + Weak expression of TREM2 by microglia is consistent with low levels of Aβ uptake. + OPN - The uptake of Aβ by microglia may also reflect engagement of the αVβ3 receptor by milk fat globule EGF factor 8 (MFG-E8), which binds to phosphatidylserine (PtdSer) molecules that may decorate Aβ plaques and promote microglial endocytosis. 3、34、35 Without wishing to be bound by theory, it is believed that MFG-E8-αVβ3 interaction is mediated by CD11c + OPN - Promotes Aβ uptake by microglia, but competition with OPN is not sufficient for CD11c + OPN + Reduced MFG-E8 binding by DP microglia and pathogenic CD11c + OPN+ Enhances TNF-α production by microglial subsets.

[0224] Taken together, our findings suggest that OPN-deficient CD11c expressing high levels of TREM2 + Our results support the view that microglia are a protective subset that actively ingests Aβ for lysosomal digestion without inducing an inflammatory response. In contrast, without wishing to be bound by theory, OPN-producing CD11c cells are unable to effectively ingest Aβ. + The (DP) microglial subset generates a robust inflammatory response that promotes AD pathology. These findings are consistent with the DAM phenotype (CD11c + This will deepen understanding of DAM 2 The protective contribution of CD11c as defined herein + OPN - These results indicate that the activity of a subset of the cells is reflective of the expression of the IL-1 receptor agonist, ... and IL-1 receptor agonist.

[0225] Colonna et al. demonstrated that TREM2 plays an essential role in regulating microglial interaction with Aβ plaques, possibly by inducing microglia to surround and transform Aβ plaque structures into a more compact morphology, thereby limiting neurite damage. 36、37 Microglial Aβ compaction depends in part on a TREM2-dependent phagocytic pathway that internalizes Aβ into activated lysosomes for digestion. 38 As a result, TREM2 deletion in both mouse models and AD patients can lead to increased Aβ burden, diffuse plaques, dystrophic neurites, and cognitive impairment. 17、26、36、39 A contribution of TAM receptors, which may act downstream of TREM2 in this plaque processing pathway, arises from the observation that TAM receptor-deficient APP / PS1 mice exhibit reduced numbers of dense-core plaques and increased cognitive impairment. 3 OPN-dependent inhibition of Aβ phagocytosis and suppression of this TREM2-lysosomal phagocytosis pathway were observed in our CD11c +Transcriptome analysis of microglia showed OPN-dependent downregulation of key components in this pathway (Trem2, Axl, Mertk, C68, and Ctsb) and OPN-deficient CD11c + We demonstrated increased microglial expression of TREM2 and CD68. The substantial reduction in plaque burden in OPN-deficient 5XFAD mice reflects accelerated lysosomal degradation of Aβ, as judged by upregulation of microglial cathepsin B and reduced diffuse plaques with increased compact plaques.

[0226] To date, clinical trials of anti-Aβ antibodies have shown that reduction in plaque burden is not accompanied by improvement in cognitive function. Recent evidence has shown that microglial deposition of previously ingested and compacted neurotoxic Aβ fibrils, via exocytosis or microglial death, is essential for the formation of non-toxic dense-core plaques that correlate with rescued cognitive function. 3、40 Without wishing to be bound by theory, it has been suggested that the packing of microglia into dense plaques of Aβ fibrils may be a neuroprotective mechanism. 3 Therefore, antibodies targeting Aβ may disassemble condensed, non-toxic Aβ fibrils into oligomers that increase neurotoxicity. 41 Thus, without wishing to be bound by theory, indiscriminate degradation of dense plaques does not efficiently ameliorate neurotoxicity and associated cognitive impairment. 42、43 Without wishing to be bound by theory, targeting OPN is a more effective therapeutic strategy than the current clinical approach of indiscriminately targeting plaques in mAb trials, as OPN deletion increases plaque compaction, substantially reduces dystrophic neurites, and improves cognitive function. In contrast, OPN deletion favorably alters the ratio of proinflammatory to phagocytic microglia, increases TREM2-associated Aβ uptake, and increases the conversion of neurotoxic Aβ material into non-toxic dense-core plaques.

[0227] Although not all findings from AD mouse models are applicable to human AD, our major findings appear to apply to the human disease. Upregulation of the gene encoding OPN (Spp1) was associated with increased oxidative stress in AD mouse models. 2、7、44 and patients 8、45、46 Interestingly, AD patients harboring TREM2 loss-of-function mutations (TREM2-R47H and TREM2-R62H) 44 However, no increase in microglial Spp1 was observed. Without being bound by theory, genetic disruption of the TREM2 pathway precludes the requirement for OPN-dependent inhibition of the TREM2 pathway in the development of plaque-associated pathology.

[0228] Increased levels of OPN in CSF and plasma samples from AD patients 47 However, the relationship between brain OPN levels and disease severity in AD patients is unclear. Consistent with our preclinical findings in 5XFAD mice, brain OPN levels, especially CD11c + OPN + The proportion of microglia strongly correlates with neuritic plaque assessment by postmortem evaluation of brain samples from AD patients. Analysis of human brain samples from well-characterized AD patients and controls also provides evidence that brain OPN levels directly correlate with disease severity by CDR score. Furthermore, CD11c + OPN + The percentage of microglia increases with the progression and severity of dementia and is strongly correlated with CDR scores. CD11c is expressed by MCI and AD patients. + OPN + Increased percentage of microglia correlates with disease onset and CD11c + We show a sensitive indicator of microglial OPN production that correlates strongly with both loss and severity of AD neuropathology. Neurofibrillary tangles, consisting mainly of aggregated hyperphosphorylated tau protein, are another key pathological hallmark of AD. We show that brain OPN levels as well as CD11c + OPN +We noted that the percentage of microglia correlated closely with the neurofibrillary tangle assessment, indicating that OPN contributes to tauopathy. These findings indicate that OPN simultaneously regulates multiple disease-spreading factors that contribute to AD pathology (i.e., neuritic plaques and neurofibrillary tangles). Without wishing to be bound by theory, the correlation of results in AD mouse models and AD patients indicates that OPN is a promising therapeutic target that may translate successfully to the clinic.

[0229] Taken together, this report distinguishes pathogenic subsets of microglia from protective microglia by expression of CD11c and production of OPN. Without wishing to be bound by theory, CD11c + OPN + Microglia are a pathogenic subset that produce substantial amounts of TNF-α and express low levels of TREM2, whereas CD11c + OPN - The microglial subset is protective because it efficiently takes up Aβ without concomitant TNF-α production and expresses high levels of TREM2. Without wishing to be bound by theory, it is possible that this OPN-producing CD11c expression may mediate the protective TREM2-lysosomal phagocytosis pathway, including promoting a proinflammatory response and inhibiting the protective TREM2-lysosomal phagocytosis pathway. + The pathogenic effects of microglial subsets reduce plaque spread and toxicity. As summarized above, CD11c, which was first defined in the murine 5XFAD model, + The levels of OPN-producing microglia positively correlate with disease severity and neuropathology in AD patients, therefore targeting microglial OPN responses may be a more effective therapeutic strategy than current antibody-based approaches that target both forms of amyloid plaques.

[0230] method mouse C57BL / 6(B6) and B6.Cg-Tg(APPSwFlLon, PSEN1 * M146L *L286V)6799Vas / Mmjax(5XFAD) mice were obtained from the Jackson Laboratory (MMRRC). flstop (OPN-KO) mice were previously generated by our laboratory. 15 Spp1 flstop Mice were crossed with 5XFAD mice to generate OPN-KO.5XFAD mice. Age-matched and gender-balanced mice were used. All mice were housed under pathogen-free conditions. All experiments were performed in accordance with federal law and institutional guidelines approved by the Dana-Farber Cancer Institute (DFCI) Animal Care and Use Committee.

[0231] Isolation of microglia Cell isolation by magnetic activated cell sorting (MACS). Adult mice were anesthetized with isoflurane and subsequently perfused transcardially using ice-cold PBS. Brains were then minced and subjected to enzymatic dissociation using collagenase type IV (300 U / mL, Worthington) or papain (20 U / mL, Worthington) and DNase I (60 U / mL, Worthington). Myelin was removed by a 30% Percoll gradient. Pelleted cells were then resuspended using MACS buffer (PBS pH 7.2, 2 mM EDTA and 0.5% BSA). Anti-mouse CD11b microbeads (Miltenyi) were used for magnetic isolation of the total microglial population. Microglia were isolated for all experiments using this method unless otherwise stated. Anti-mouse ACSA-2 microbeads (Miltenyi) were used for isolation of astrocytes. Neurons were negatively enriched using anti-CD11b, anti-ACSA-2 and anti-O4 microbeads (Miltenyi) to deplete microglia, astrocytes or oligodendrocytes.

[0232] Microglia isolation and fluorescence-activated cell sorting for RNA-seq analysis. Single cell suspensions were prepared as previously described. 9、48Briefly, mice were anesthetized with isoflurane and perfused transcardially using ice-cold PBS. Brains were rapidly excised and minced using a scalpel on ice, followed by Dounce homogenization in ice-cold Hank's Balanced Salt Solution (HBSS). Cell suspensions were passed through a 70 μm cell strainer and then spun down at 500 g for 5 min at 4 °C. Myelin and debris were removed using 10 mL of ice-cold 40% Percoll (Sigma) and spun at 500 g for 30 min, followed by washing with 10 mL of ice-cold HBSS and spinning again at 500 g for 5 min at 4 °C. All samples were then resuspended in ice-cold FACS buffer (0.5% BSA, 1 mM EDTA in 1x PBS) for staining. Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and anti-CD16 / CD32 antibodies (2.4G2, 1:100, BD Biosciences) were used to exclude dead cells and block Fc receptors, respectively. Single cell suspensions were then stained with anti-CD11b (M1 / 70, 1:100, Biolegend), anti-CD45 (30-F11, 1:100, Biolegend) and anti-CD11c (N418, 1:50, Biolegend) antibodies for 20 min on ice. Samples were then washed with ice-cold FACS buffer and spun down at 500g for 5 min. Cell pellets were resuspended in 5 mL of FACS buffer and then sorted on a BD FACS Aria II using a 70 μm nozzle in purity mode and a sorting speed of approximately 10,000 events / s. After sorting, each sample was spun down and the cell pellet was immediately stored at -80°C until further processing. For 9-month-old 5XFAD mice, duplicate CD11c pooled from 44 mice (22 males + 22 females) were + Microglia and CD11c - Microglia were sequenced. Duplicate CD11c pooled from 27 mice (13 males + 14 females) for 9-month-old OPN-KO.5XFAD mice. + Microglia were sequenced.

[0233] Flow cytometry Flow cytometry analysis of microglia. Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and anti-CD16 / CD32 antibody (2.4G2, 1:100, BD Biosciences) were used to exclude dead cells and avoid non-specific staining, respectively. Microglia were stained with appropriate surface markers, such as anti-CD11b (M1 / 70, 1:100 Biolegend), anti-CD45 (30-F11, 1:100 Biolegend), anti-CD11c (N418, 1:50 Biolegend), anti-αV integrin (RMV-7, 1:50, Biolegend), anti-β3 integrin (2C9.G2, 1:50, Biolegend), anti-β5 integrin (KN52, 1:50, eBioscience), anti-CD44 integrin (IM7, 1:100, BD Bioscience), anti-TREM2 (237920, 1:20, Novus Biologicals), followed by anti-OPN (IC808, 1:10, R&D Biosciences). Cells were fixed and permeabilized with an Intracellular Fixation and Permeabilization Buffer Kit (eBioscience) for subsequent intracellular staining with anti-TNF-α (MP6-XT22, 1:50, Biolegend) and anti-TNF-α (MP6-XT22, 1:50, Biolegend). Data were acquired on a CytoFLEX LX (Beckman Coulter) and analyzed with FlowJo (Tree Star).

[0234] Validation of microglial CD11c expression. Validation of microglial CD11c expression was performed as previously described. 9Briefly, single-cell brain suspensions from 9-month-old 5XFAD mice were stained with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and anti-CD16 / CD32 antibodies (2.4G2, 1:100, BD Biosciences), followed by incubation with anti-CD11b (M1 / 70, 1:100, Biolegend), anti-CD45 (30-F11, 1:100, Biolegend), anti-CD11c (N418, 1:50, Biolegend), anti-TMEM119 (106-6, 1:200, ABCAM), and anti-CCR2 (SA203G11, 1:100, Biolegend). + Cells were gated from single / live cells and subsequently identified as microglia with CD11b + CD45 low and CD11b as macrophages + CD45 high To distinguish between microglia and macrophages, we used the microglia-specific marker Tmem119, which is expressed by blood-derived macrophages but not by microglia. 49 and CCR2 50、51 CD11b expressing CCR2 but not Tmem119 + CD45 high The cells were identified as macrophages, while all expressed Tmem119 but not CCR2, CD11b + CD45 low The cells are identified as microglia. + CD45 low To confirm the specificity of CD11c staining in microglial populations, we included a fluorescence minus one (FMO) negative control. Brain CD45 contains primarily non-immune cells that do not express CD11c. - Cells were also included as negative controls to further verify the staining specificity.

[0235] Real-time quantitative PCR (RT-qPCR). Microglial RNA was extracted using the RNeasy Plus Universal Mini Kit according to the manufacturer's instructions (QIAGEN). Complementary DNA (cDNA) was then reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using 100 ng of RNA according to the manufacturer's instructions. Real-time quantitative PCR of Spp1 was performed using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems) with 5 μl of cDNA, 4.92 μl of PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) and 0.08 ul of primers (IDT, working concentration: 200 nM) per reaction. Normalized to β-actin ΔΔ The gene expression levels of Spp1 were compared using the Ct method.

[0236] water t maze Animal cognition testing was performed at the NeuroBehavior Laboratory at the Harvard Institute of Medicine. The water T-maze (WTM) behavioral paradigm assesses spatial learning and spatial memory by training mice to navigate to a hidden platform using spatial cues in a room to escape from the water. This test also measures cognitive flexibility by a reversal learning procedure in which mice must learn the new location of the hidden platform. Testing was performed as previously described. 52The testing apparatus was a plus-maze (each arm 14 cm long and 4.6 cm wide) made of clear plexiglass, with each arm termed North (N), South (S), East (E), or West (W). A divider was placed on the maze to block the appropriate arm so that the mouse could only choose the E or W arm for escape. At the start of each trial, mice were placed in the N or S arm in a semi-random order. The maze was filled with water (25–26 °C) and the escape platform was placed on the E side of the maze submerged approximately 1 cm below the surface of the water. The water was made opaque by adding white non-toxic paint to prevent the mouse from seeing the submerged platform. At the start of a trial, a divider was placed in place to block the appropriate arm, and the mouse was carried to the appropriate starting point. The experimenter scored a correct or incorrect response for each trial, and the mouse was allowed to stay on the platform for 10 s before being removed. Mice were given 10 trials per day, and the accuracy rate was calculated by averaging the accuracy rate over the 10 trials on each day. The platform was then moved to the opposite side, and the same procedure was repeated for reversal trials until the mice learned the new location of the platform.

[0237] RNA-seq analysis of CD11c microglia RNA extraction. Total RNA was extracted from FACS-sorted cell pellets using the RNeasy Plus Universal Mini Kit according to the manufacturer's instructions (QIAGEN). Library preparation, sequencing reactions and bioinformatic analysis (gene hit counts) were performed at GENEWIZ, LLC. (South Plainfield, NJ, USA) as follows.

[0238] Library preparation. The concentration of extracted RNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was confirmed using an Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). CD11c RNA obtained from adult mice was + Because microglial cell numbers are very limited, microglial samples from 9-month-old 5XFAD and OPN-KO.5XFAD mice were processed with the SMART-Seq v4 Ultra Low Input Kit (Clontech, Mountain View, CA) for full-length cDNA synthesis and amplification for sequencing, and Illumina Nextera XT libraries were used for sequencing library preparation. Briefly, cDNA was fragmented using transposase and adapters were added, followed by limited cycle PCR to enrich and index the cDNA fragments. The final libraries were evaluated on an Agilent TapeStation.

[0239] HiSeq sequencing. Sequencing libraries were clustered on flow cell lanes. After clustering, the flow cells were loaded into an Illumina HiSeq instrument 4000 according to the manufacturer's instructions. Samples were sequenced using a 2 × 150 bp paired-end (PE) configuration. Image analysis and base calling were performed with HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from the Illumina HiSeq were converted to fastq files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

[0240] RNA-Seq data analysis. Mapping and gene counting were performed by GENEWIZ (South Plainfield, NJ, USA). After checking the quality of the raw data, sequence reads were trimmed to remove low-quality adapter sequences and nucleotides using Trimmomatic v.0.36. The trimmed reads were mapped to the reference genome of Mus musculus available at ENSEMBL using STAR aligner v.2.5.2b. STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the whole read sequence. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature counts from the Subread package v.1.5.2. Only unique reads that fall within exon regions were counted. Significant differentially expressed genes (DEGs) were then detected using a negative binomial model implemented in the R package edgeR. 53、54 Gene expression was considered upregulated if log2FC>0.5, and downregulated if log2FC<-0.5. DEGs were considered significant with a false discovery rate (FDR) adjusted p-value <0.05. FC represents the fold change in reads per million kilobases (RPKM) at FDR <0.05.

[0241] Immunofluorescence staining Mouse brains were removed after transcardial perfusion and fixed in 4% paraformaldehyde solution (PFA, Electron Microscopy Sciences) overnight at 4° C. After rinsing with PBS, the fixed brains were dehydrated in 30% sucrose overnight at 4° C. The brains were then embedded in OCT compound (Sakura Finetek) and serial sagittal frozen sections (10 μm) were cut using a Cryostat (CM3050S, Leica).

[0242] Brain cryosections were permeabilized with PBS containing 0.1% Triton X100 (PBS-T) for 1 h, followed by incubation in blocking solution (Jackson ImmunoResearch Lab) containing 5% normal donkey serum in PBS-T for 1 h to prevent nonspecific binding. Sections were then incubated for 24 h with the appropriate primary antibodies: rabbit anti-Iba-1 (1:1000, WAKO), anti-amyloid beta (6E10, 1:1000, Biolegend), biotin anti-mouse CD11c (N418, 1:50, Biolegend), goat anti-OPN (AF808, 1:100, R&D), anti-APP (A4, 1:1000, Millipore), sheep anti-TREM2 (AF1729, 1:10, R&D), rat anti-CD68 (FA-11, 1:100, Bio-Rad), and goat anti-cathepsin B (AF965, 1:100, R&D). To amplify the CD11c signal, sections were washed with PBS and incubated for 5 min with the Alexa Fluor 488 streptavidin Tyramide SuperBoost Kit (Invitrogen) according to the manufacturer's instructions. Sections were then incubated for 1 h with the appropriate secondary antibody: Alexa-594-conjugated donkey anti-rabbit IgG or Alexa-488-conjugated donkey anti-mouse IgG (1:500; Invitrogen). Brain cryosections incubated without anti-CD11c primary Ab or tyramide signal amplification (TSA) are included as negative controls to exclude nonspecific immunofluorescence signals. DAPI (Invitrogen) was used as nuclear counterstain (10 min incubation). For Thioflavin S staining, brain cryosections were incubated in filtered 1% Thioflavin S aqueous solution for 10 min at room temperature and washed 2x3 min in 80% ethanol and 3 min in 95% ethanol before adding blocking solution. Samples were analyzed on an Olympus fluorescent microscope. Quantification of fluorescent microscopy images was performed using ImageJ (NIH).

[0243] In vitro microglia assay CD11c +To investigate the effect of OPN-αVβ3 interactions on microglial proinflammatory responses, microglia were enriched by anti-CD11b microbeads (>95% purity, Miltenyi) from 9-month-old OPN-KO.5XFAD mice and cultured at 3 × 10 in conventional microglial culture medium (DMEM-F12 containing 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / mL recombinant mouse M-CSF). 5 Microglia were seeded at 100 cells / well in 12-well plates. Microglia were preincubated with the selective αVβ3 inhibitor Cilengitide (10 μM) for 1 h, followed by addition of 12.5 μg / mL recombinant mouse OPN (rmOPN). After 24 h of culture, TNF-α was detected by flow cytometry using CD11c expression. + Analysis of microglial expression was performed.

[0244] To examine the effect of OPN on TREM2-lysosomal activation and Aβ degradation, anti-CD11b microbead (>95% purity, Miltenyi) enriched microglia from 9-month-old 5XFAD and OPN-KO.5XFAD mice were cultured at 3 × 10 in conventional microglial culture medium (DMEM-F12 containing 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / mL recombinant mouse M-CSF). 5 Cells / well were seeded in 12-well plates. Microglia were then preincubated with anti-OPN Ab (10 μg / mL) for 1 h, followed by incubation with rmOPN (12.5 μg / mL) overnight. FAM-labeled Aβ was then 1-42 Peptides (1 μM) were added to the cultures for 1 h. CD11c after 1 h of incubation + Microglia (CD11c + CD68 + ) FAM-Aβ in lysosomes 1-42 The mean fluorescence intensity (MFI) of Aβ was determined by flow cytometry. 1h Then, FAM-Aβ 1-42The cells were then harvested and cultured for 24 hours in the presence or absence of anti-OPN Ab (10 μg / mL) and / or rmOPN (12.5 μg / mL), and then CD11c was detected by flow cytometry. + CD68 + Microglia (AβMFI 24h FAM-Aβ retained in the 1-42 TREM2, CD68 and CD11c in MFI + Microglial expression was analyzed. Aβ degradation rate was calculated using the formula (AβMFI 1h -AβMFI 24h ) / AβMFI 1h It was calculated as:

[0245] Processing and evaluation of human brain samples Clinical and postmortem diagnosis, brain harvesting procedures, tissue processing and assessment of AD pathology have been described in detail elsewhere. 29 Clinically and neuropathologically characterized brain tissue from the middle frontal gyrus (an area affected early by AD) in AD patients and controls. 29 were obtained from the Mount Sinai Brain Bank through Neurobiobank (https: / / neurobiobank.nih.gov / ). Three groups of deceased subjects were included in this study: cognitively normal (n=11), mild cognitive impairment (MCI, n=10), and AD patients (n=11). Tables 2 and 3 describe the cohort in detail. Cognitive status at the time of death was confirmed using the Clinical Dementia Rating (CDR) scale. 55 The CDR assesses cognitive and functional impairments associated with dementia and provides specific severity criteria for classifying subjects as non-demented (CDR=0), suspected dementia (CDR=0.5), or increasing levels of dementia severity from CDR=1 to CDR=3. Participants with acute neurological conditions such as stroke or traumatic brain injury were excluded.

[0246] Evaluation of plaques and tangles in human brain. Evaluation of neuronal plaques and neurofibrillary tangles was performed at the Mount Sinai brain bank according to the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) protocol. 56 Sections from paraffin-embedded blocks were variously stained with hematoxylin and eosin, modified Bielschowsky, modified thioflavin S, as well as anti-β-amyloid (4G8), anti-tau (AD2). All neuropathological data regarding the extent and distribution of neuropathological lesions were collected in a blinded fashion with respect to the dementia status of the subjects. Each case was assigned a Braak AD staging score for the progression of neurofibrillary neuropathology. 57、58 In addition, quantitative data regarding neuritic plaque density was collected as described. 30 .

[0247] Human OPN ELISA. For quantification of human brain OPN concentration, 10 mg of human brain frozen tissue was homogenized in 0.3 mL of lysis buffer [20 mM Tris-Hcl pH 8, 130 mM NaCl, 1% Triton X100 and protease inhibitor cocktail (Roche)], kept on ice for 45 min and centrifuged at 13000 g for 20 min at 4°C. Protein concentration of brain lysates was measured using a BCA kit (ThermoFisher Scientific) and 10 μg of protein was loaded into each well of a human OPN Quantikine ELISA plate (R&D Systems). The procedure was performed according to the manufacturer's instructions.

[0248] CD11c + OPN +Human brain staining of microglia. Paraffin-embedded brain tissue sections from the middle frontal gyrus were deparaffinized in xylene, rehydrated in gradient ethanol, and washed in PBS containing 0.1% Triton X100 (PBS-T). Brain sections were boiled in 10 mM citrate buffer (pH 6) for antigen retrieval and washed in PBS-T. Endogenous peroxidase activity of the samples was quenched with 3% hydrogen peroxide solution for 60 min, washed, and incubated with Zyblack (Zytovision, BS-0002-8) for 30 min to reduce autofluorescence. Endogenous biotin was blocked using Biotin-Blocking Kit (Invitrogen, E21390). Staining was performed by multiplexing three Tyramide SuperBoost Kits (Invitrogen, B40936, B40912, B40923). First, sections were blocked with blocking buffer for 60 min, then incubated with primary antibodies [biotinylated anti-OPN 1:50 (R&D Systems, BAF1433), anti-CD11c 1:150 (Novus, NBP2-44598) and anti-Iba-1 1:500 (Wako, 019-19741)] at 4°C overnight. After washing, sections were incubated with HRP-conjugated streptavidin for 60 min, washed, incubated with Alexa Fluor™ 647 Tyramide streptavidin reagent for 10 min, and then incubated with stop solution for 5 min. This was followed by Superboost kits Alexa Fluor™ 488 tyramid anti-mouse and Alexa Fluor™ 555 tyramid anti-rabbit. Cell nuclei were marked with Hoechst, and slides were mounted with Immu-Mount medium (Thermofisher Scientific).

[0249] Brain sections from control (n=5), MCI (n=9) and AD (n=8) were coded and captured under a confocal microscope (Leica DMi8) with the same laser intensities, 10-12 images in triplicate, at a magnification of X400, with laser 408 nm at 5% exposure, laser 488 nm at 0.1% exposure, laser 638 nm at 6.5% exposure and laser 552 nm at 3% exposure. Triple positive cells expressing Iba-1, CD11c and OPN, as well as total Iba-1 positive cells in each slice, were counted using ImageJ software (NIH). CD11c among total Iba-1 positive cells was counted. + OPN + Microglia (CD11c + OPN + Iba-1 + ) was calculated for each section and the mean value was calculated for each brain sample. Analyses were performed by a blinded investigator.

[0250] Statistics and Reproducibility Data are presented as mean ± sem. Statistical analysis was performed using GraphPad Prism software version 9.0. Analysis for comparison of multiple groups was performed by one-way or two-way ANOVA with Bonferroni test. Comparison between two groups was analyzed by two-tailed Student's t test. Correlation analysis was performed using Pearson correlation. P<0.05 was considered significant. * P < 0.05, ** P < 0.01, *** P<0.001, **** P<0.0001. Statistical details such as the specific statistical tests used in the different analyses as well as the respective sample sizes are given in the respective figure legends.

[0251] Data availability CD11c + Microglia, CD11c from 9-month-old 5XFAD mice - Microglia and CD11c from 9-month-old OPN-KO.5XFAD mice +RNA-seq data for microglia have been deposited in the NCBI-Gene Expression Omnibus (GEO) under the accession number GSE191118. The data are available upon reasonable request from the authors.

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[0253] Example 3 Example 3 -Characterization of Angiopep-2 Ab Antibodies have been modified by many methods to address the bottlenecks in their delivery to various tissues, but penetrating the blood-brain barrier (BBB) ​​currently remains the most challenging obstacle. One approach is based on the conjugation of Angiopep-2 peptides to host proteins, such as immunoglobulins, to increase their penetration through the endothelial lining of the brain. In principle, receptor-dependent transcytosis of Angiopep-2 (through its interaction with low-density lipoprotein receptor-related protein 1 [LRP-1]1) could facilitate the passage of its cargo through the BBB. However, in our hands, this method was not satisfactory in terms of its impact on brain penetration and on the ability of transcytosinated antibodies to bind antigens in the brain.

[0254] Although several patents use Angiopep-2 to enhance the penetration of small molecules or multiple peptides into the brain, we could only identify one patent (US Pat. No. 9,161,988) that uses Angiopep-2 Ab conjugates to enhance BBB penetration. We tested this method according to brain penetration by tagged Angiopep-conjugated Abs and the antigen binding capacity of the penetrated Abs (neither of these issues are addressed in the above-mentioned US Pat. No. 9,161,988). We noted a relatively weak activity (FIG. 21). We reasoned that modifying the positive charge of Angiopep2-Ab could enhance its interaction with the negatively charged endothelial cells lining the brain. Without wishing to be bound by theory, increasing the flexibility of the Angiopep-2-Ab conjugation bridge reduces its aggregation potential and makes the conjugated Ab more functional. These considerations led to an approach to this problem that embodies the features described below. Briefly, we designed a KK modification and an Ahx linker to enhance brain penetration of Angiopep2-conjugated mAbs.

[0255] First, we add two lysines (-kk-, pI 9.74) to the bridge between Ab and angiopep-2 to increase the isoelectric point (pI) of the conjugated antibody (to alkaline levels). Without wishing to be bound by theory, this enhances penetration into the BBB via adsorption-mediated transport (AMT), which relies on interactions between the negatively charged surface of endothelial cells and positively charged displaced ligands.

[0256] Second, we add an Ahx linker to the Angiopep2 sequence, which increases the flexibility of the peptide chain and allows it to be properly solvated to prevent aggregation and the associated loss of receptor-mediated transcytosis (RMT) and functional penetration by Angiopep-2 Ab (Figure 22).

[0257] References cited in this example Demeule, M. et al. Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2. J Neurochem 106, 1534-1544, doi:10.1111 / j.1471-4159.2008.05492.x (2008).

[0258] Example 4 Example 4 - Development of an artificial brain-penetrating monoclonal antibody (mAb) targeting osteopontin (OPN) for the treatment of Alzheimer's disease Recent clinical trials of anti-amyloid-β (Aβ) antibodies have reported significant reductions in plaque burden without appreciable cognitive improvement. These disappointing results have accelerated efforts to identify and validate new drug targets that may halt or reverse disease progression and cognitive decline. Alternative approaches are illustrated by recent division of Aβ plaques into two subsets: a diffuse invasive subset of neurotoxic plaques that impair cognition, and a dense subset that may represent a non-toxic product of microglial processing and compaction of Aβ fibrils (Huang et al., 2021; Parhizkar et al., 2019; Yuan et al., 2016). If microglial packing into dense plaques of Aβ fibrils is a neuroprotective mechanism, generalized disassembly of dense plaques by antibodies may be counterproductive. These considerations also indicate that therapeutic enhancement of microglial plaque compaction may be a more effective treatment strategy than the current approach of indiscriminately targeting plaques in mAb trials.

[0259] Based on preclinical studies using the 5XFAD model of Alzheimer's disease (AD) and the analysis of a panel of well-characterized human brain tissues, we have defined the Spp1 gene (encoding osteopontin, OPN) as a new tractable target. We also developed a method to test the therapeutic potential of a blood-brain barrier (BBB)-penetrating antibody specific for OPN. This anti-OPN mAb could improve cognitive function in 5XFAD mice by reducing neurotoxic diffuse Aβ plaques and microglial inflammatory responses.

[0260] Defining OPN as a therapeutic target for AD: Although many disease-associated genes have been identified in mouse models of AD, most have not been confirmed from human AD experiments. The Spp1 gene (encoding OPN) is a notable exception, as it appears to be strongly upregulated by microglia in both animal models of AD and human disease. Our recent studies show that genetic deletion of OPN in 5XFAD mice substantially reduces inflammatory microglia, amyloid beta (Aβ) plaque areas, and significantly improves cognitive function. Our mechanistic studies show that targeting OPN derepresses TREM2-dependent pathways involved in microglial uptake, lysosomal digestion, and extrusion of Aβ fibrils into the brain parenchyma (Huang et al., 2021; Keren-Shaul et al., 2017; Parhizkar et al., 2019; Yuan et al., 2016), thereby reducing the spread of toxic Aβ oligomers throughout the brain.

[0261] We have tested the relevance of these mouse findings in human disease using clinically and neuropathologically characterized brain tissue from AD patients and controls (Mt.Sinai Brain Bank). OPN production by human microglia correlates closely with both AD severity and pre- and post-mortem assessments of cognitive impairment, as judged from comparisons of brain material from AD patients and healthy controls (in collaboration with Dr. Michal Beeri, Mt.Sinai). Increased numbers of OPN-producing microglia in AD brain slices correlate closely with progressive dementia, as measured by the Clinical Dementia Rating (CDR) scale. Based on these studies, blocking OPN may reduce pro-inflammatory microglia and promote lysosomal degradation and detoxification of associated Aβ plaques, thereby improving cognitive function.

[0262] Generation and testing of monoclonal anti-OPN Abs designed to enhance brain penetration: We generated a fluorophore-labeled (AF488) mAb after conjugation of a monoclonal anti-OPN Ab (MPIIIB10) with a chemically modified Angiopep-2 peptide (Ang2pep). Ang2pep mediates receptor-dependent transcytosis and crossing of the blood-brain barrier (BBB) ​​via interaction with low-density lipoprotein receptor-related protein 1 (LRP-1) expressed by brain endothelial cells. This strategy allows for OPN blockade for therapeutic purposes in vivo.

[0263] What are the distinguishing features of the disclosed approach? What improvements or advantages does the disclosed approach offer over existing approaches? Our approach is based on a more precise definition of the contribution of OPN, which has been identified as part of the genetic signature in both AD and animal models of human disease. Monoclonal Ab targeting of OPN may reduce Aβ pathology and improve cognitive function, in contrast to current Aβ-based approaches that indiscriminately target all Aβ plaques. We designed a brain-penetrant anti-OPN mAb to selectively target neurotoxic Aβ plaques as a proof-of-principle experiment and as a basis for the development of novel and effective antibody-based AD therapies.

[0264] Several features of OPN increase the clinical feasibility of targeting this protein with mAbs. OPN is an extracellular protein secreted by microglia that is easily accessed by mAbs. OPN production is generally associated with pathogenic rather than defensive responses. Expression of OPN in peripheral tissues contributes to several chronic disorders such as atherosclerotic and cardiovascular diseases, autoimmune diseases, and cancer growth and metastasis, and genetic or antibody-based reduction of OPN ameliorates pathology in these clinical settings. In contrast, gene deletion or reduction of OPN does not suppress general immune responses.

[0265] What applications are envisioned for the disclosed techniques? How would those applications work? OPN expression substantially increases proinflammatory microglia and impairs lysosomal degradation and associated detoxification of Aβ plaques. Efficient targeting of OPN with anti-OPN mAbs is applicable to AD and other OPN-mediated neuroinflammatory diseases, such as multiple sclerosis (MS).

[0266] Because OPN expression is upregulated in microglia during disease progression and the continuation of AD, antibody neutralization of OPN is during the time window when OPN-mediated effects are most destructive and pathogenic.

[0267] What other techniques can be used in combination with those disclosed herein? The following procedure can be used to further enhance anti-OPN mAb brain penetration.

[0268] 1. Cell-penetrating peptides (CPPs) are a group of short peptides consisting of amphipathic and / or cationic sequences that allow them to cross cell membranes. For example, transactivator of transcription peptide (TAT) is an effective CPP that has been found to effectively transport various therapeutic reagents to the brain in mouse models with various CNS disorders. Positively charged TAT can promote interaction with negatively charged cell membranes at the BBB and induce internalization via adsorptive-mediated transcytosis (AMT). This is a receptor / transporter-independent pathway that is distinct from the Ang2pep pathway. If necessary, we will construct an Ang2pep-TAT dual conjugate mAb to see whether Ab conjugates utilizing both receptor-mediated transcytosis (RMT) and adsorptive-mediated transcytosis (AMT) pathways would allow increased penetration and higher Ab concentrations in brain tissue.

[0269] 2. A two-step injection regimen can be used to maximize brain penetration of anti-OPN mAbs. Unconjugated ("cold") anti-OPN mAb can be injected at time 0, allowing for occupancy of available OPN and Fc receptors expressed by peripheral tissues prior to injection of Angiopepe2-conjugated ("hot") anti-OPN mAb, thus enhancing brain penetration of this conjugated "hot" Ab.

[0270] Further research plans are described.

[0271] We found that genetic deletion of OPN in the 5XFAD mouse model inhibits microglial production of inflammatory cytokines and promotes TREM2-dependent microglial uptake of amyloid fibrils and associated lysosomal activation, resulting in increased condensation and extrusion of non-toxic Aβ plaques. These changes are accompanied by a marked improvement in cognitive function. We designed a brain-penetrating peptide-conjugated anti-OPN mAb to examine its therapeutic efficacy in vivo using a mouse model of AD. Analysis of the brain penetration and therapeutic activity of the conjugated anti-OPN mAb relies on a comparison between OPNWT.5XFAD and OPN- / -.5XFAD mice to distinguish specific anti-OPN binding to brain OPN from non-specific uptake of anti-OPN mAb (MPIIIB10).

[0272] To analyze brain penetration of anti-OPN mAbs, brains and major peripheral organs (spleen, kidney, liver, etc.) of 5XFAD and OPN- / -.5XFAD mice injected with conjugated or unconjugated Abs are harvested and the intensity of the AF488 fluorophore is measured using a fluorescence microplate reader to obtain raw fluorescence units (RFU). Antibody concentration is quantified as percent of injected dose per gram of tissue (%ID / g): %ID / g = measured RFU per gram of brain (or tissue) / total injected RFU. Furthermore, a brain capillary depletion assay is performed to distinguish Ang2pep-mediated transcytosis of Abs into brain parenchyma from binding to brain microvasculature according to the (AF488) intensity of the fluorophore in capillary-enriched versus parenchymal fractions of brain homogenates at different time points after Ab injection. The binding specificity of the conjugated Ab at the optimized dose is also confirmed by counterstaining of OPN brain sections from 5XFAD and OPN- / -.5XFAD mice with a second fluorophore after injection of the conjugated Ab.

[0273] The therapeutic activity of the conjugated anti-OPN mAb using an optimized dosing regimen is evaluated for its impact on Aβ plaque pathology and neuroinflammation according to Aβ plaque number / area, plaque compaction and number of TNF-α+ microglia. Aβ plaque burden is examined by immunofluorescence of hippocampal and cortical sections with anti-Aβ Ab 6E10 and confirmed by Aβ positron emission tomography (PET) imaging according to mean PET standardized uptake value ratio (SUVR) composite images of the score 18F-Florbetapir tracer that binds to Aβ plaques. Lysosomal activation of microglia (CD68 lysosomal activating protein and cathepsin B enzyme) is evaluated by immunofluorescence of mouse brain cryosections. The impact of anti-OPN mAb on neuropathology is examined according to the number of dystrophic neurites with labeled anti-APP Ab and, if necessary from the above analysis, is further confirmed by testing of cognitive function using the water T-maze and novel Y-maze.

[0274] References cited in this Example. Huang, Y., Happonen, KE, Burrola, PG, O'Connor, C., Hah, N., Huang, L., Nimmerjahn, A., and Lemke, G. (2021). Microglia use TAM receptors to detect and engulf amyloid beta plaques. Nat Immunol. Keren-Shaul, H., Spinrad, A., Weiner, A., Matcovitch-Natan, O., Dvir-Szternfeld, R., Ulland, TK, David, E., Baruch, K., Lara-Astaiso, D., Toth, B., et al. (2017). A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease.Cell 169,1276-1290 e1217. Parhizkar, S., Arzberger, T., Brendel, M., Kleinberger, G., Deussing, M., Focke, C., Nuscher, B., Xiong, M., Ghasemigharagoz, A., Katzmarski, N., et al. (2019). Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE.Nat Neurosci 22,191-204. Yuan, P., Condello, C., Keene, CD, Wang, Y., Bird, TD, Paul, SM, Luo, W., Colonna, M., Baddeley, D., and Grutzendler, J. (2016). TREM2 Haplodeficiency in Mice and Humans Impairs the Microglia Barrier Function Leading to Decreased Amyloid Compaction and Severe Axonal Dystrophy. 92,252-264.

[0275] Example 5 Example 5 - Development of an artificial brain-penetrating monoclonal antibody (mAb) targeting osteopontin (OPN) for the treatment of Alzheimer's disease An exemplary method for developing an artificial brain-penetrant monoclonal antibody to target OPN for the treatment of Alzheimer's disease is provided below.

[0276] In working models, OPN inhibition of microglial Aβ plaque compaction and promotion of inflammatory responses contribute to cognitive impairment. The definition of OPN as a therapeutic target for Alzheimer's disease (AD) comes from i) data from the 5XFAD mouse model, and ii) data from AD patients and controls (Mount Sinai brain bank). Additionally, monoclonal anti-OPN Abs were designed to enhance brain penetration.

[0277] An exemplary working model of microglial OPN expression is shown in Figure 23. In this model, OPN expression promotes microglial pro-inflammatory responses (TNF-α) and inhibits the TREM2 / Axl / lysosomal phagocytosis pathway, thereby suppressing lysosomal degradation and Aβ plaque compaction, which ultimately leads to cognitive impairment. The OPN-based platform described herein targets diffuse Aβ plaques and the microglial pro-inflammatory response, simultaneously inhibiting two disease-increasing factors.

[0278] Microglia are the major cellular source of OPN production in the brains of 5XFAD mice. As shown in Figure 24, microglial expression of OPN increased at both the mRNA and protein levels in 5XFAD mice compared to age-matched WT mice as the disease progressed.

[0279] OPN expression is upregulated by CD11c in the brains of 5XFAD mice + Restricted to microglia. CD11c + OPN + The percentage of microglia increased substantially in 5XFAD mice compared to age-matched WT mice during disease progression (Figure 25).

[0280] Genetic depletion of OPN reduced microglial TNF-α production, total Aβ plaque area, diffuse plaque percentage and number of atrophic neurites, and rescued cognitive impairment (FIG. 26).

[0281] CD11c + Transcriptome analysis of microglia revealed that OPN deletion resulted in downregulation of proinflammatory response-related genes and upregulation of phagocyte-related genes. + CD11c + Reduced TNF-α production by microglia, but not αVβ3 - CD11c +It did not reduce TNF-α production by microglia, indicating that OPN promotes microglial proinflammatory responses through its interaction with the αVβ3 integrin receptor.Furthermore, in situ analysis of brain cryosections from 5XFAD mice showed that OPN deficiency increased microglial phagocytosis of Aβ (Figure 27).

[0282] TREM2 upregulates CD11c in the brains of 5XFAD mice. + It is expressed exclusively by microglia. Genetic deletion of OPN reduced the expression of CD68 and cathepsin B, CD11c, in OPN-KO.5XFAD mice compared to 5XFAD mice. + As judged by increased microglial expression, CD11c + OPN upregulates CD11c expression in 5XFAD mice, resulting in increased microglial TREM2 expression and lysosomal activation. + We demonstrated that it inhibits the TREM2-lysosomal phagocytosis pathway in microglia (Figure 28).

[0283] Significant upregulation of plaque compaction index (6E10 + Thio-S + Area / 6E10 + As judged by area, OPN deletion resulted in a substantial reduction in the total area of ​​Aβ plaques (6E10 + ) and increased compact plaque area (6E10 + Thio-S + ) and OPN recruits CD11c + The results show that it inhibits microglial compaction (Figure 29).

[0284] Measurement of OPN in middle frontal gyrus homogenates revealed a three-fold increase in OPN expression in the brains of AD patients compared to OPN expression in cognitively normal control subjects. Increased OPN expression in the brains of AD patients compared to the brains of MCI patients was also observed, but did not reach statistical significance. Correlation analysis of brain OPN expression and CDR scores showed that increased OPN expression was positively correlated with dementia severity (Figure 30).

[0285] We determined the percentage of CD11c+OPN+ (double positive, DP) microglia by immunofluorescence analysis of human brain sections of the middle frontal gyrus, an area affected early by AD. Normal controls and MCI subjects showed similar percentages of CD11c+OPN+ microglia (CD11c+OPN+Iba-1+). We found a three-fold increase in CD11c+OPN+ microglia in brain sections of AD patients compared to sections of normal controls. Furthermore, the percentage of CD11c+OPN+ microglia from AD patients was significantly higher than that from MCI patients, indicating that the percentage of CD11c+OPN+ microglia is a sensitive parameter for the discrimination of AD patients from those suffering from MCI. This conclusion is further supported by the very strong correlation of the percentage of CD11c+OPN+ microglia with the CDR score (Figure 31).

[0286] Higher brain OPN levels correlated with higher neuritic plaque levels (r=0.4919, p=0.0043). Similar to the association seen with dementia severity, there was a strong correlation between the percentage of CD11c+OPN+ (DP) microglia and the density of neuritic plaques (r=0.8226, p<0.0001). These findings further support the view that the percentage of CD11c+OPN+ microglia may be a sensitive marker of both clinical severity and neuropathology of AD (Figure 32).

[0287] We conjugated Angiopep2 to anti-CD11b mAb and tested the binding activity of the conjugated and unconjugated mAb. After incubation with microglia from 5XFAD mice, Angiopep2-conjugated and unconjugated anti-CD11b mAb showed similar binding activity (AF488+ microglia) and mean fluorescence intensity (MFI), indicating that conjugation does not change the binding activity of the mAb (Figure 33).

[0288] We conjugated a modified Angiopep2 peptide (containing a -KK- amino acid bridge and an AhX linker) to an anti-CD11b mAb. In vivo studies demonstrated efficient brain penetration of the conjugated anti-CD11b mAb. The Angiopep-2 conjugated Ab showed a 10-fold increase over the non-conjugated anti-CD11b Ab (Figure 34).

[0289] Microglia isolated from 9-month-old 5XFAD mice were incubated with increasing concentrations (5, 10, 20 μg / mL) of anti-OPN mAb (MPIIIB10) for 24 h, followed by CD11c + Flow cytometric analysis of TNF-α production by microglia was performed. Microglia incubated with isotype control (mouse IgG1) were included as a negative control. Blocking anti-OPN mAb inhibited CD11c + This resulted in a dose-dependent inhibition of TNF-α production by microglia (Figure 35).

[0290] Example 6 Example 6 - Exemplary Approaches to Treating Alzheimer's Disease Our approach to the treatment of Alzheimer's disease is based in part on the following; Unmet needs: i) Dementia / Alzheimer's disease affects 55,000,000 people (2020) with a projected 130,000,000 affected in 2050; ii) no effective treatment; anti-Aβ antibodies (e.g., aducanumab) reduce plaque without cognitive improvement ($55,000 / year); and iii) the impact of long-term care on families, healthcare systems and society.

[0291] OPN-based therapeutics: engineered brain-penetrant anti-OPN mAb selectively removes toxic plaques and improves cognitive function.

[0292] A schematic (Figure 36) shows OPN-dependent regulation of protective Aβ degradation and compaction by microglia. In OPN-low brains, microglia engulf and degrade Aβ plaques in activated lysosomes and subsequently extrude non-toxic compacted plaques into the microenvironment. In OPN-high AD brains, OPN inhibits microglial engulfment and compaction of toxic diffuse plaques while promoting a proinflammatory response (TNF-α induction). OPN blockade with anti-OPN mAb or integrin inhibitors inhibits the microglial proinflammatory response and enhances microglial engulfment and compaction of diffuse plaques (Figure 36).

[0293] The schematic in Figure 37 depicts an exemplary OPN mechanism of action. We demonstrate that pathogenic OPN-producing CD11c kinase promotes proinflammatory responses and inhibits microglial engulfment / compaction of Aβ plaques, thereby contributing to AD pathology and cognitive impairment (Figure 37). + Microglial subsets were identified.

[0294] Genetic deletion of OPN results in the formation of neurotoxic plaques (6E10 + Thio-S - ) and a reduction in the number of dystrophic neurites, which ultimately rescued 5XFAD mice from cognitive impairment (Figure 38).

[0295] In a mouse model of AD (5XFAD mice), OPN deficiency resulted in a substantial reduction in toxic diffuse plaques and an increased expression of pathogenic CD11c + OPN + These findings were validated in AD patients using clinically and neuropathologically defined brain samples. + OPN + The percentage of microglia was highly correlated with the Clinical Dementia Rating (CDR) scale, neuritic plaque score and tau tangle score (Figure 39).

[0296] We conjugated a brain-crossing Ab containing the Angiopep2 peptide, a -KK-amino acid bridge and an AhX linker to an anti-CD11b mAb. In vivo studies demonstrated efficient brain penetration of the brain-crossing conjugated anti-CD11b mAb. The Angiopep2-conjugated Ab showed approximately a two-fold increase in fluorescent microglia compared to the unconjugated anti-CD11b Ab. The KK-Ahx-modified Angiopep-2 Ab showed a two- to three-fold increase over the Angiopep-2-conjugated Ab and a ten-fold increase over the unconjugated anti-CD11b Ab (Figure 40).

[0297] The first generation of brain-penetrating conjugates of anti-OPN mAbs involved the conjugation of modified Angiopep2 (containing a -KK- bridge and an AhX linker) with anti-OPN mAbs. Another conjugation strategy can generate an Angiopep2-TAT double conjugate that can further increase the brain penetration of anti-OPN mAbs (Figure 41).

[0298] Example 7 Example 7 - Administration of anti-OPN mAb inhibits microglial proinflammatory responses and ameliorates Aβ plaque pathology Weekly administration of anti-OPN to 5XFAD mice was continued for 1 or 2 months at 6 months of age. Microglial proinflammatory responses and Aβ plaque pathology were analyzed after 1 or 2 months of treatment (Figure 42A).

[0299] One month of treatment resulted in a modest reduction, and two months of treatment resulted in a 45% reduction in CD11c+ microglia compared to control mice. One month of treatment resulted in a 35% reduction, and two months of treatment resulted in a 55% reduction in TNF-α expression by CD11c+ microglia compared to isotype-matched control mice (Figure 42B).

[0300] Representative immunofluorescence images of 5XFAD mouse brains after 1 or 2 months (FIG. 42C and FIG. 42D). The first row of images in (C) and (D) were stained with 6E10 to identify both diffuse and condensed forms of Aβ plaques. The second row of images were stained with thioflavin-S to identify β-sheet structures. + Only Aβ condensed plaques were identified. Column 3 shows a merged image of columns 1 and 2. Quantification of total plaque area, diffuse plaque area and compactness index of plaques after 1 and 2 months of treatment is shown in (E-G).

[0301] Example 8 Example 8 - Administration of cyclic RGD (Cilengitide) inhibits microglial pro-inflammatory responses Weekly doses of cyclic RGD (Cilengitide) were administered intravenously or intranasally to 6-month-old mice for 1 or 2 consecutive months. Microglial proinflammatory responses and Aβ plaque pathology were analyzed after 1 or 2 months of treatment (FIG. 43A).

[0302] Intranasal cilengitide resulted in a reduction in CD11c+ microglia at 1 month and a 35% reduction at 2 months compared to control mice.Intranasal cilengitide resulted in an approximately 35% reduction in TNF-α expression by CD11c+ microglia at 1 month and an approximately 45% reduction at 2 months compared to control mice (Figures 43B and 43C).

[0303] Intravenous cilengitide resulted in a 50% reduction in CD11c+ microglia at 1 month and a 60% reduction at 2 months compared to control mice. Intravenous cilengitide resulted in approximately a 50% reduction in TNF-α expression by CD11c+ microglia at 1 month and a 55% reduction at 2 months compared to control mice (Figures 43B and 43C).

[0304] Example 9 Example 9 - Intranasal Administration of Antibodies In general, the administration of Angiopep-2 conjugated antibodies in many of the experiments described above was by intravenous route.Without wishing to be bound by theory, osteopontin is present in multiple tissues, so these tissues can act as "sinks" for anti-OPN antibodies when the antibodies are administered systemically (e.g., intravenously).

[0305] In the experiments described in this example, intranasal administration of conventional antibodies (i.e., antibodies that are not conjugated to a moiety such as Angiopep-2 for delivery of an active / therapeutic agent) was tested as a way to increase delivery of the antibody to the brain, presumably avoiding the "sink" effect that occurs with intravenous administration of Angiopep-2 conjugated antibodies. Without wishing to be bound by theory, intranasal delivery was tested as a way to circumvent the blood-brain barrier by using olfactory transduction to deliver agents to the brain.

[0306] Figures 44A-C show intranasal delivery of conventional non-conjugated anti-CD11b monoclonal antibody. Intranasal delivery of the antibody (10 mg / kg body weight) to 6-month-old 5XFAD mice (n=4) was performed at time 0. The antibody was labeled with the fluorophore AF488. Observations were performed 3 hours after administration. The control was an intravenous injection of anti-CD11b monoclonal antibody (also non-conjugated). (A) Anti-CD11b monoclonal antibody fluorescence in the brain (green) for intranasal administration vs. control intravenous administration. (B) Anti-CD11b monoclonal antibody fluorescence in the brain (green) for intranasal administration, also showing immunofluorescence of anti-Iba-1 antibody (red). Iba-1 is a microglial marker. The merged immunofluorescence (yellow) confirms the binding specificity of anti-CD11b monoclonal antibody to microglia. (C) Data for calculation of anti-CD11b monoclonal antibody penetration into the brain in these experiments are presented as the number of fluorescent microglia per microscopic field (μm) for anti-CD11b monoclonal antibody administered via the intranasal route compared to intravenous administration. 2 ×10 6) The data show that intranasal administration of the antibody resulted in approximately a 10-fold increase in brain penetration compared to intravenous injection.

[0307] Example 10 Example 10 - OPN affects inflammasome activation in microglia Activation of the NLRP3 (NLR family pyrin domain containing 3) inflammasome is a major neuroinflammatory component of Alzheimer's disease (AD) pathology in both human and mouse models of AD. We analyzed the effect of OPN on inflammasome activation in microglia in vitro according to caspase-1 activity and IL-1β production. Microglial intracellular caspase-1 activity was measured using a bioluminescence assay that specifically detects caspase-1 activation and confirmed by a selective caspase-1 inhibitor (Figure 45A). We found that recombinant mouse OPN (rmOPN) substantially increased caspase-1 activity and IL-1β production in LPS+Aβ-stimulated microglia of both 9-month-old 5XFAD and OPN-KO.5XFAD mice. This OPN-dependent promotion of inflammasome activation was blocked by an αVβ3 inhibitor (Figures 45B-C), indicating that OPN-dependent promotion of microglial inflammasome activation may reflect its interaction with αVβ3 integrin.

[0308] In these experiments, microglia isolated from 9-month-old 5XFAD and OPN-KO.5XFAD mice were cultured at 6 × 10 4Cells / well were seeded in 96-well plates. To induce inflammasome activation, microglia were primed with 100 ng / mL LPS for 3 h and subsequently stimulated overnight with 10 μM Aβ1-42 fibrils in the presence or absence of 12.5 μg / mL rmOPN. An αVβ3 inhibitor (Cilengitide, Selleck, 10 μM) was added to the cultures 1 h prior to the addition of rmOPN. Microglial intracellular caspase-1 activity was analyzed by the bioluminescent method Caspase-Glo® 1 Inflammasome Assay Kit (Promega) according to the manufacturer's instructions. The detection specificity of caspase-1 activity was verified using a selective caspase-1 inhibitor (Ac-YVAD-CHO, 1 μM) included in the kit. Culture media was collected and microglial IL-1β production was quantitatively measured by Mouse IL-1β DuoSet ELISA Kit (R&D Systems) according to the manufacturer's instructions.

[0309] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures specifically described herein which equivalents are considered to be within the scope of this invention and are covered by the following claims.

[0310] Table 1. DEGs in CD11c+ microglia in 9-month-old OPN-KO.5XFAD vs. 5XFAD TIFF2025508682000003.tif221129TIFF2025508682000004.tif228128TIFF2025508682000005.tif229128TIFF2025508682000006.tif228128TIFF2025508682000007.tif229128TIFF2025508682000008.tif229128TIFF2025508682000009.tif229128TIFF2025508682000010.tif228128TIFF2025508682000011.tif228128TIFF2025508682000012.tif228128TIFF2025508682000013.tif228128TIFF2025508682000014.tif228128TIFF2025508682000015.tif228128TIFF2025508682000016.tif228128TIFF2025508682000017.tif229128TIFF2025508682000018.tif228128TIFF2025508682000019.tif228128TIFF2025508682000020.tif229128TIFF2025508682000021.tif229128TIFF2025508682000022.tif228128TIFF2025508682000023.tif229128TIFF2025508682000024.tif228128TIFF2025508682000025.tif229128TIFF2025508682000026.tif228128TIFF2025508682000027.tif229128TIFF2025508682000028.tif229128TIFF2025508682000029.tif228128TIFF2025508682000030.tif228128TIFF2025508682000031.tif228128TIFF2025508682000032.tif228128TIFF2025508682000033.tif228128TIFF2025508682000034.tif228128TIFF2025508682000035.tif229128TIFF2025508682000036.tif228128TIFF2025508682000037.tif228128TIFF2025508682000038.tif229128TIFF2025508682000039.tif228128TIFF2025508682000040.tif229128TIFF2025508682000041.tif229128TIFF2025508682000042.tif228128TIFF2025508682000043.tif228128TIFF2025508682000044.tif229128TIFF2025508682000045.tif229128TIFF2025508682000046.tif228128TIFF2025508682000047.tif228128TIFF2025508682000048.tif228128TIFF2025508682000049.tif229128TIFF2025508682000050.tif229128TIFF2025508682000051.tif229128TIFF2025508682000052.tif228128TIFF2025508682000053.tif228128TIFF2025508682000054.tif228128TIFF2025508682000055.tif229128TIFF2025508682000056.tif228128TIFF2025508682000057.tif228128TIFF2025508682000058.tif229128TIFF2025508682000059.tif229128TIFF2025508682000060.tif228128TIFF2025508682000061.tif228128TIFF2025508682000062.tif229128TIFF2025508682000063.tif229128TIFF2025508682000064.tif229128TIFF2025508682000065.tif228128TIFF2025508682000066.tif228128TIFF2025508682000067.tif228128TIFF2025508682000068.tif229128TIFF2025508682000069.tif228128TIFF2025508682000070.tif208128.

[0311] (Table 2) Summary of human samples included in the study. TIFF2025508682000071.tif45170Age- and sex-matched samples were analyzed for OPN levels measured by ELISA on frozen samples followed by immunofluorescence staining for Iba-1 / CD11c / OPN (fixed paraffin-embedded sections).

[0312] (Table 3) Characterization of frozen and fixed human samples. TIFF2025508682000072.tif250170

Claims

1. A compound capable of crossing the blood-brain barrier of a patient, conjugated to a crosslinking moiety, linker, carrier agent, or combination thereof; anti-osteopontin antibody, anti-CD11b antibody, or αVβ3 inhibitor; The compound comprises an active agent.

2. 2. The compound of claim 1, wherein the αVβ3 inhibitor comprises a cyclic RGD-containing small molecule, including cilengitide or a derivative thereof.

3. The compound of claim 1 , wherein the anti-osteopontin antibody or anti-CD11b antibody comprises a monospecific antibody, a bispecific antibody, or a multispecific antibody.

4. 2. The compound of claim 1, wherein the anti-osteopontin antibody or anti-CD11b antibody comprises a monoclonal antibody, Fab, F(ab')2, Fab' single chain antibody, Fv, single chain, monospecific antibody, bispecific antibody, trispecific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, shark antibody, nanobody, camelid antibody, microbody, intrabody, defucosylated antibody, or any combination or derivative thereof.

5. The compound of claim 1 , wherein the anti-osteopontin antibody or anti-CD11b antibody comprises a single-chain antibody.

6. The compound of claim 1 , wherein the carrier agent comprises a cell-penetrating peptide or a cell-targeting peptide.

7. 7. The compound of claim 6, wherein the carrier agent comprises TFFYGGSRGKRNNFKTEEY (Angiopep-2; SEQ ID NO: 1), D-Lys6-LHRH (SEQ ID NO: 3), CNGRCG (SEQ ID NO: 4), PGA,LHRH (SEQ ID NO: 5), DRDDS (SEQ ID NO: 6), D-γ-E-γ-E-γ-E-E (SEQ ID NO: 7), GSH,HSTPSSP (SEQ ID NO: 8), DSSLFAL (SEQ ID NO: 9), YGRKKRRQRRRPPQQ (SEQ ID NO: 10), LLIILRRRRIRKQAHAHSK (SEQ ID NO: 11), RRLSYSRRRF (SEQ ID NO: 12), or any combination thereof.

8. The compound of claim 1 , wherein the crosslinking moiety comprises a positively charged amino acid.

9. 9. The compound of claim 8, wherein the linking moiety comprises arginine, lysine, histidine, or a combination thereof.

10. The compound of claim 1 , wherein the linker comprises an amino acid comprising a flexible linker.

11. The compound of claim 10 , wherein the flexible linker comprises a small, polar or non-polar amino acid.

12. 11. The compound of claim 10, wherein the flexible linker comprises 6-aminohexanoic acid (Ahx), 2-aminoethoxyacetic acid (AEA), 5-aminovaleric acid (Ava), 8-amino-3,6-dioxaoctanoic acid (PEG2 or AEEA), or 12-amino-4,7,10-trioxadodecanoic acid (PEG3).

13. The compound of claim 1 further comprising a conjugate that connects the active agent to the bridge, linker, or carrier agent.

14. The compound of claim 13 , wherein the conjugate comprises an —N-hydroxysuccinimide ester (—NHS ester).

15. The compound of claim 1, having a linear arrangement of an active agent, a bridging moiety, a linker, and a carrier agent in that order.

16. A pharmaceutical composition for treating a neurodegenerative disease in a patient, comprising a compound according to any one of claims 1 to 15.

17. A pharmaceutical composition for treating Alzheimer's disease in a patient, comprising a compound according to any one of claims 1 to 15.

18. 18. The pharmaceutical composition of claim 17, which is for intranasal administration.

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