Compositions and methods for treating neuropathy

Abstract

The present invention relates to engineered brain penetrating therapeutic compounds and methods of use thereof. Methods of intranasal administration of active agents are also disclosed.

Description

[0001] Related Applications

[0002] This application claims priority or the benefit of U.S. Provisional Application No. 63 / 306,985 filed on February 4, 2022, U.S. Provisional Application No. 63 / 347,113 filed on May 31, 2022, and U.S. Provisional Application No. 63 / 480,398 filed on January 18, 2023 under 35 U.S.C. 119, the contents of each of which are incorporated herein by reference in their entirety.

[0003] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein.

[0004] 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 files and records, but otherwise reserves any and all copyrights whatsoever.

[0005] Government interests

[0006] This invention was made with government support under Grant No. R01048125 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0007] The present invention relates to engineered brain-penetrating therapeutic compounds and methods of use thereof. Background Art

[0008] Alzheimer's disease (AD) is a progressive neurological disease that causes brain shrinkage and brain cell death. AD is the most common cause of dementia, a persistent decline in thinking, behavior, and social skills that affects a person's ability to live independently. Worldwide, approximately 55 million people currently suffer from dementia, and this number is expected to rise to 78 million by 2030 and 139 million by 2050. There is no effective treatment to cure AD or to alter the disease process in the brain. Summary of the invention

[0009] In general, the present disclosure relates to compounds that can cross the blood-brain barrier (BBB) ​​of a patient. These compounds can contain a "payload" or active agent, which can be a therapeutic agent, a cytotoxic agent, an imaging agent, etc. These compounds typically contain a portion for delivering the active agent across the BBB. In an embodiment, the portion can include one or a combination of a carrier agent, a bridge (e.g., an amino acid bridge), and a linker (e.g., a flexible linker).

[0010] One aspect of the invention relates to a therapeutic compound that can cross the blood-brain barrier of a patient. In embodiments, 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 embodiments, 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.

[0011] Another aspect of the invention includes a moiety for delivering a therapeutic agent across the blood-brain barrier of a patient. In certain embodiments, the moiety comprises 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 by receptor-dependent transcytosis (RDT). In embodiments, the amino acid bridge is configured to cross the blood-brain barrier via adsorption-mediated transcytosis (AMT).

[0012] 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.

[0013] In various aspects and embodiments disclosed herein, the flexible linker comprises 6-aminohexanoic acid (Ahx).

[0014] In certain aspects and embodiments disclosed herein, the carrier agent comprises Angiopep-2.

[0015] The therapeutic agent may comprise an anti-osteopontin (OPN) antibody. The antibody may be conjugated to a moiety for delivering the therapeutic agent across the blood-brain barrier. In some embodiments, the antibody may be conjugated to the moiety via MFCO-N-hydroxysuccinimide ester.

[0016] Additional aspects include a pharmaceutical composition for treating a neurodegenerative disease in a patient, the pharmaceutical composition comprising an anti-osteopontin (OPN) antibody. In embodiments, the pharmaceutical composition comprises a component for crossing the blood-brain barrier. In embodiments, neurodegenerative diseases include Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, etc. In certain embodiments, the component for crossing the blood-brain barrier comprises any of the various embodiments described herein with respect to the section.

[0017] In another aspect, the present invention includes a method of delivering a medicament to the central nervous system of a patient in need thereof. In some embodiments, the method includes administering a medicament (e.g., a therapeutic agent or moiety as described herein) peripherally and allowing the medicament to pass through the blood-brain barrier. In some embodiments, the medicament can be administered intravenously. In some embodiments, the medicament can be delivered to the brain. In some embodiments, the activating agent can be an antibody.

[0018] 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, the intranasal delivery of the active agent may not include a portion for delivering the agent across the blood-brain barrier. In some embodiments, the intranasal delivery of the active agent may result in delivery to the brain without passing through the blood-brain barrier. In some embodiments, the intranasal delivery of the active agent may result in olfactory transfer of the active agent to the brain. In some embodiments, the active agent may be an antibody.

[0019] Yet another aspect includes a method of treating a neurodegenerative disease in a patient. In embodiments, the method includes administering to the patient any pharmaceutical composition disclosed herein. Neurodegenerative diseases may include Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and other diseases. In embodiments, the means for crossing the blood-brain barrier comprises any of the various embodiments described herein with respect to the part.

[0020] In certain embodiments, the therapeutic compound comprises Fig. 22 structure.

[0021] Other objects and advantages of the present invention will become apparent from the ensuing description.

[0022] Other objects and advantages of the present invention will become apparent from the ensuing description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A to 1F show CD11c + Microglia express CD11c when they are engulfed by apoptotic neurons (AN) in early development. -(A) Generation of brain single-cell suspensions from 9-month-old WT mice for verification of microglial CD11c expression by flow cytometry. We first expressed CD11b + Cells were gated and subsequently screened for CD11b + The CD45 low population was gated. Almost all cells in this population (about 99%) were Tmem119 + , while CCR2 expression was undetectable, suggesting that CD11b + The CD45 low population is microglia. In contrast, the CD11b + CCR2 but not Tmem119 expression was detected in the CD45 high fraction (i.e., a putative macrophage population). FMO negative controls for CD11b were used in these FACS plots. + Analysis of the CD45 low microglia population confirmed specificity. Brain CD45-, which contains mainly non-immune cells (neurons, astrocytes, oligodendrocytes, but not microglia) that do not express CD11c, was used as a negative control to further validate the specificity of this FACS strategy. (B) CD11c expression in C57BL / 6 (B6) mice (n=3) during early development and normal aging was determined by flow cytometric analysis. + The proportion of microglia.CD11c + Microglia are initially present in late embryonic stages (E18.5), peak in early post-anal age (P5), and gradually decline to marginal levels during young adulthood (3 months), followed by reappearance and further expansion during aging. (C) CD11c were isolated from P5B6-WT mice by - Microglia: Negative sections were treated with anti-CD11c magnetic beads (purity > 99%) and then incubated for 72 hours in the presence or absence of apoptotic neurons (AN) labeled with pHrodo fluorescent dye at a 1:1 ratio. - 23% of microglia took up AN (AN + ), where these AN + 77.4% of microglia are CD11c + In contrast, CD11c - <1% of the total microglia (AN-, which reflects 77% of the total microglia in 72-hour culture) expressed CD11c (upper panel). These findings suggest that after incubation with AN, total CD11c - About 18% of microglia express CD11c +, whereas 98% of microglia incubated for 72 h in the absence of AN retained CD11c - (Bottom). (D, E) CD11c isolated from P5B6-WT mice - Microglia were incubated in the presence or absence of LPS (10 ng / ml) or Aβ (1 μM) for 24 h, followed by FACS analysis of microglial activation markers and CD11c expression. + MHCII + CD11c expression was assessed in activated microglia. Although CD86 and MHCII were upregulated in response to LPS and Aβ, CD11c was not observed in response to these stimuli. - Microglial differentiation. FACS plots are representative data from 3 experiments. (F) CD11c cells were isolated from P5B6-WT mice by - Microglia: Negative sections were treated with anti-CD11c magnetic beads (purity > 99%) and then incubated for 72 h with or without the αVβ3 integrin inhibitor Cilengitide (Ci1) (10 μM) and / or the pan-TAM receptor inhibitor LDC1267 (1 μM) in the presence or absence of pHrodo fluorescent dye-labeled AN at a 1:1 ratio. - Microglial uptake of AN was reduced by approximately 50%, and the combination of the two inhibitors further suppressed AN uptake to background levels. Within 72 h, stimulation of AN induced approximately 20% of the initial CD11c - Microglia expressed CD11c, whereas in the absence of AN stimulation, CD11c expression was undetectable during the same period. Inhibition of AN uptake by cilengitide or LDC1267 reduced CD11c - CD11c + The acquisition of the phenotype was reduced by approximately 50% to 75% in each case, and the addition of both inhibitors completely prevented this phenotypic shift (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 shown as mean±sem.

[0024] Figure 2Validation of microglial OPN expression by flow cytometry is shown. Microglial OPN expression was validated in 9-month-old WT mice by flow cytometric analysis 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) at 4°C for 30 minutes. Isotype controls (1:10, PE-conjugated goat IgG) and OPN-KO microglia were used as negative controls. Microglia that selectively express an intracellular isoform of OPN (OPN-i-KI) were used as positive controls. WT microglia and OPN-i-KI microglia showed similar levels of OPN staining, while staining of microglia from OPN-KO donors or microglia stained with isotype controls did not produce detectable signals.

[0025] Figures 3A to 3H show that the stability of microglial expression of CD11c is regulated by OPN. (A) CD11c isolated from B6-WT mice at different stages of development and aging + CD11c - Flow cytometric analysis of OPN expression in microglia (n=3) OPN production is restricted to CD11c during early development and normal aging + Microglia. Two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001. (B) CD11c - Microglia were isolated from P5B6-WT mice and then co-incubated with pHrodo fluorescent dye-labeled apoptotic neurons (AN) at a 1:1 ratio for 72 h. - Differentiation of microglia into CD11c + Microglia, accompanied by a sharp increase in OPN expression (n = 3). ****p < 0.0001 by two-tailed Student's t-test. (C, D) CD11c + Microglia (>95% purity) were isolated from P5 and 9-month-old WT and OPN-KO mice and then cultured in regular microglial culture medium (DMEM-F12 with 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / ml M-CSF) for 7 days. + The percentage of microglia was evaluated by CD11c + In vitro analysis of microglial stability (percentages normalized to day 0). CD11c in P5WT mice and 9-month-old WT mice + Microglia are stable, while 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 multiple comparison test. (E, F) Evaluation of CD11c using isolated organotypic hippocampal slice cultures (OHSCs) + Microglial stability. Freshly prepared OHSCs were incubated with 0.5 mg / ml clodronate liposomes at 35 °C for 24 h to deplete endogenous microglia. 4 × 10 isolated from P5 or 9-month-old WT or OPN-KO mice were used. 3 CD11c + Microglia supplemented each OHSC without microglia (purity>95%). Representative images showing the expression of P5WT or P5OPN-KO CD11c + CD11c in microglial remodeling of hippocampal slices + Microglia. CD11c in P5WT and 9-month-old WT mice + Microglia stably express CD11c, while OPN deficiency reduces CD11c expression by 60% (n=3-4). Scale bar: 25 μm. ****p<0.0001, ***p<0.001 by two-way ANOVA with Bonferroni multiple comparison test. (G, H) CD11c isolated from 9-month-old WT and OPN-KO mice + Microglia were incubated in vitro or in OHSCs in the presence of 1 μM synthetic human Aβ 1-42 peptide for 7 days. CD11c from 9-month-old WT mice + Microglia were stable, while OPN deficiency resulted in a significant reduction of 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 are shown as mean±sem.

[0026] Figure 4A-4B shows that OPN deficiency reduces CD11c + (A) Flow cytometry analysis of CD11c in WT and OPN-KO mice during early development and aging. + and CD11c - OPN deficiency resulted in a decrease in the proportion of CD11c in P5, 6-month-old, and 9-month-old WT mice +The proportion of microglia was significantly reduced. An isotype control of CD11c was used as a negative control (n=3). **p<0.01, ****p<0.0001 by two-way ANOVA with Bonferroni multiple comparison test. (B) Analysis of CD11c in brain cryosections of WT and OPN-KO mice at P5 and 9 months of age + and CD11c - The proportion of microglia in OPN-KO mice showed CD11c + The proportion of microglia was significantly reduced (n=3). + (CD11c + Iba-1 + ) or CD11c - Microglia (CD11c - Iba-1 + ) in total microglia (Iba-1 + ) to evaluate the percentage of CD11c + CD11c - Proportion of microglia. Brain slides incubated without anti-CD11c primary antibody 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.

[0027] Figures 5A-5B show that CD11c + The number of microglia was comparable in WT and OPN-KO mice. (A, B) CD11 c was compared between P5 and 9-month-old WT mice and age-matched OPN-KO mice at day 0 by flow cytometry (in vitro) and immunofluorescence staining (in OHSCs). + Number of microglia in vitro and in OHSCs, day 0 CD11c + The number of microglia was similar between P5, 9-month-old WT mice and OPN-KO mice (n=3).Statistical analysis was performed by two-tailed Student's t-test.

[0028] Figure 6 Validation of CD11c expression by flow cytometry on microglia subjected to cold separation is shown. Brain single cell suspensions of 9 month old WT mice were generated by cold separation and used to validate microglial CD11c expression. + Cells were gated on single cells / live cells and CD11b + CD45 低 Cells were gated as microglia and CD11b+ CD45 高 Gated as macrophages. CD11b + The majority of cells (95%) in the CD45 low population were Tmem119 + , while CCR2 expression was undetectable, suggesting that CD11b + The CD45 low population represents microglia. In contrast, the CD11b + CD45 高 CCR2 but not Tmem119 was detected in macrophages. + Analysis of the CD45 low microglial population confirmed the specificity of CD11c staining. - cells (containing mainly non-immune cells that do not express CD11c) were included as a negative control.

[0029] 7A to 7G show CD11c + Definition of the intrinsic gene program of microglia. (A) Analysis of CD11c by RNA-seq in P5 and 9-month-old WT mice + and CD11c - Transcriptome profiling of microglia. Venn diagram showing the transcriptome of WT CD11c cells at P5 and 9 months of age. + and CD11c - Genes expressed in microglia and the number of genes exclusively expressed by each microglial subset. - CD11c was identified among the genes negatively expressed in the counterparts + Microglial unique genes. Based on the expression of CD11c - The raw counts of genes not expressed in microglia (e.g., Itgax) were used to define the threshold for negative expression. A similar strategy was used to identify CD11c - Microglia-specific genes. (B) CD11c + Core gene signature of microglia identified as CD11c at P5 and 9 months of age + (C) Heat map showing the overlap of unique genes in microglia with CD11c - Compared with the counterparts, P5 and 9-month-old CD11c + Four CD11c in microglia +Microglial core genes, including Itgax, Cd209a, Cd209f, and Cd36 (FDR < 0.05). (D, E) Validation of core genes at the protein level. Validation of CD11c at the protein level by flow cytometry analysis + Core genes of microglia. CD11c - Compared with microglia, the expression of surface proteins CD36 and CD209a was only increased by CD11c in P5WT mice and 9-month-old WT mice. + Microglial cell expression (n=3). ****p<0.0001, **p<0.01 by two-tailed Student's t-test. (F) CD11c isolated from P5WT mice - Microglia were incubated with LPS (10 ng / ml) or Aβ (1 μM) for 24 h, followed by flow cytometric analysis of CD36 and CD209 expression. + Activated CD11c - Protein expression of these core genes was barely detectable in microglia. FACS graphs are representative of 3 experiments. (G) CD11c + In vitro differentiation of microglia. Freshly isolated CD11c from P5WT mice + and CD11c - Microglia. CD11c was validated by analyzing mRNA expression of representative core genes at day 0 + Microglial phenotype. Then CD11c - Microglia were incubated with apoptotic neurons (AN) for 72 h to induce CD11c - Differentiation of microglia into CD11c + Microglia. CD11c by Q-PCR on day 3 + Microglia (differentiated) and CD11c - Core genes validated in microglia (undifferentiated). - Compared with microglia at day 0, CD11c + CD11c expression in microglia + Microglial phenotype was indicated by upregulation of selected core genes. AN-induced CD11c - Microglial differentiation is accompanied by CD11c + Induction of microglial gene program. The expression of core genes was normalized to z-scores and shown in the heat map (n=3), P<0.05.

[0030] Figures 8A to 8G show that OPN regulates CD11c +Intrinsic functions of microglia. (A) Microglia isolated from P5 and 9-month-old WT, OPN-KO, and OPN-i-KI mice were compared with 1×10 5 136 mg pHrodo Green-labeled synaptosomes were incubated for 1 hour and then CD11c + Flow cytometric analysis of microglial phagocytosis of synaptosomes.CD11c from P5 but not 9-month-old WT mice + Microglia have a strong ability to phagocytose synaptosomes in vitro. The lack of OPN-s significantly abolished this function, while OPN-i was not involved in this process (n=3). One-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001. (B) Ex vivo analysis showed that CD11c from P5WT mice but not 9-month-old WT mice + Microglia display robust activity in phagocytosis of synaptic proteins. P5WT mice lacking OPN-s but not OPN-i display greatly reduced CD11c + Phagocytosis of presynaptic protein synaptophysin or postsynaptic protein PSD95 by microglia (n=3). ****p<0.0001, ***p<0.001 by two-tailed Student's t-test. (C) CD11c + Microglia express high levels of the proliferation marker Ki-67 early in development (P5). Lack of OPN-s but not OPN-i reduces CD11c + Ki-67 expression in microglia (n=3). One-way ANOVA with Bonferroni multiple comparison test, *p<0.05. (D) During senescence, CD11c + Microglia showed relatively low levels of proliferative activity, and in mice lacking OPN-s, CD11c + Ki-67 expression in microglia was significantly reduced (n=3). *WT vs. OPN-KO, #WT vs. OPN-i-KI. Two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, *p<0.05, #### p<0.0001, # p<0.05. (E) 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 the proliferation marker Ki-67. rmOPN enhanced CD11c + αVβ3 +Ki-67 expression in microglia was significantly inhibited by αVβ3 inhibitor (n=3). **p<0.01, *p<0.05 by one-way ANOVA with Bonferroni multiple comparison test. (F) Expression of TNF-α in CD11c+ Tgp11 cells of P5 mice + It is barely detectable in microglia, but its expression increases during aging. + Age-dependent increase in TNF-α expression in 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. Two-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001, #### p<0.0001. (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. + αVβ3 + In microglia, the upregulation of TNF-α expression induced by rmOPN was completely abolished by αVβ3 inhibitor (n=3). **p<0.01 by one-way ANOVA with Bonferroni's multiple comparison test. Data are shown as mean±sem.

[0031] Fig. 9 Figure 2 shows that microglial αVβ3 expression gradually increases 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 increases during aging. Representative plots from 4 independent experiments.

[0032] 10A to 10D show CD11c + Microglial core gene program and OPN-dependent stable phenotype are preserved in the context of 5XFAD disease. (A, B) Validation of selected CD11c at the protein level by flow cytometry + Compared with CD11c microglia, protein expression of CD209a and CD36 was only increased in CD11c microglia of 9-month-old 5XFAD mice. + Microglial expression. Isotype controls of CD36 and CD209a were used as negative controls. (n=3). ****p<0.0001 by two-tailed Student's t-test. (C) CD11c isolated from 9-month-old 5XFAD or OPN-KO.5XFAD mice+ Microglia were cultured in vitro or in OHSC for 7 days. CD11c in 9-month-old 5XFAD mice + Microglia are stable in vitro and in OHSCs, whereas CD11 + The stability of microglia was dramatically reduced by 40% to 80% in vitro and in OHSCs, respectively (n=3). ****p<0.0001, ***p<0.001 by two-way ANOVA with Bonferroni multiple comparison test. (D) CD11c isolated from 9-month-old 5XFAD or OPN-KO.5XFAD mice + Microglia were cultured in vitro or in OHSCs in the presence of 1 μM synthetic human Aβ1-42 peptide for 7 days. CD11c from 9-month-old 5XFAD mice + Microglia are stable, and OPN deficiency leads to CD11c + Microglial stability was acutely reduced in response to Aβ stimulation in vitro or in OHSCs (n=3-4).***p<0.001, **p<0.01 by two-way ANOVA with Bonferroni's multiple comparison test. Data are shown as mean±sem.

[0033] Figures 11A to 11L show that OPN deficiency alleviates AD pathology and rescues cognitive deficits in 5XFAD mice. (a) Microglia (CD11b + ), astrocytes (GFAP + ) and neurons (MAP2 + ) Flow cytometric analysis of OPN expression in the brain of 5XFAD mice. 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). During disease progression, microglial OPN expression in 5XFAD mice increased at both the mRNA level and the protein level compared to age-matched WT mice (n=3-4). By two-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001, ***p<0.001, *p<0.05. (d) CD11c from 5XFAD mice at different disease stages + and CD11c - Flow cytometric analysis of OPN expression in microglia. OPN production is restricted to CD11c during 5XFAD disease progression. +Microglia (n=3). Two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001. (e) Flow cytometric analysis showed that CD11c in 5XFAD mice was upregulated compared with age-matched WT mice during disease progression. + OPN + The percentage of microglia increased (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 deficiency reduced the disease-dependent increase in microglial TNF-α production in 5XFAD mice (n = 3). *WT vs. 5XFAD, ****p < 0.0001 by two-way ANOVA with Bonferroni's multiple comparison test; # 5XFAD and OPN-KO.5XFAD, #### p<0.0001. (g, h) Representative immunofluorescence images and quantification of Aβ area in brain cryosections of 5XFAD and OPN-KO.5XFAD mice at different disease stages. OPN deficiency leads to a significant reduction in total Aβ plaque area in the cortex and hippocampus of 5XFAD mice during disease progression (n=3-4). Two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, *p<0.05, ns: not significant. Scale bar = 200 μm. (i) Staining of different forms of Aβ plaques with anti-Aβ (6E10) and Thio-S in brain cryosections of 9-month-old 5XFAD and OPN-KO.5XFAD mice. OPN deficiency leads to diffuse plaques (6E10 + Thio-S - ) in total plaque (6E10 +) percentage decrease (n=3). By two-tailed Student's t-test, ***p<0.001. (j, k) Representative immunofluorescence images and quantification of dystrophic neurites (labeled with N-terminal APP) per plaque (labeled with thioflavin-S) in brain sections of 5XFAD and OPN-KO.5XFAD mice. The number of dystrophic neurites was quantified within a 25 μm area of ​​each plaque. Compared with 5XFAD mice, the number of dystrophic neurites per plaque in OPN-KO.5XFAD mice was significantly reduced (n=30 to 40 plaques, from 3 mice in each group). By two-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001, ***p<0.001, ns: not significant. Scale bar = 200 μm. (1) Spatial learning and memory (acquisition test) and cognitive flexibility (reversal test) were assessed by T-type water maze in 9-month-old 5XFAD and OPN-KO.5XFAD mice. Age-matched WT mice were induced as controls. 5XFAD mice lacking OPN showed increased percentage of correct choices in both acquisition and reversal tests (n=3-5, # :WT and 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.

[0034] Figures 12A-12B show confirmation of OPN KO in OPN-KO.5XFAD mice. (a) PCR genotyping results, which confirm the successful knockout of OPN in OPN-KO.5XFAD mice. WT ) shows 300 bp OPN WT Band. After hybridization with OPN-KO mice, the resulting OPN-KO.5XFAD mice obtained a 500bp OPN KO band. All mice showed a 377bp 5XFAD transgenic band and a 324bp internal positive control IL-2 band. (b) OPN KO was verified at the protein level in microglia of 9-month-old OPN-KO.5XFAD mice. Microglial expression of OPN was not detected in OPN-KO.5XFAD mice. Isotype controls were used as negative controls, while microglia from age-matched 5XFAD mice were used as positive control cells. The contour plots are representative results from 3 independent experiments.

[0035] Fig.13 Flow cytometry validation of CD11c expression on microglia in 5XFAD mice is shown. Flow cytometry validation of CD11c expression on microglia in 9-month-old 5XFAD mice. + Cells were gated on single cells / live cells and CD11b + CD45 低 Gating on microglia and CD11b + CD45 高 Gated as macrophages. CD11b + The majority (about 90%) of the CD45 low population are Tmem119 + , while CCR2 expression was undetectable, confirming that CD11b + CD45 低 In contrast, in the CD11b + CD45 高 CCR2 but not Tmem119 was detected in macrophages. + CD45 低 Analysis of the microglial population confirmed the specificity of CD11c staining. Brain CD45- cells (containing mainly non-immune cells that do not express CD11c) were included as a negative control.

[0036] Figures 14A to 14L show that pathogenic CD11c + OPN production by microglia promotes proinflammatory responses and inhibits Aβ uptake in the brain of 5XFAD mice. (a) Heat map showing CD11c from 9-month-old 5XFAD mice compared to OPN-KO.5XFAD mice. + Representative differentially expressed genes (DEGs) of microglia. CD11c + OPN deficiency in microglia results in downregulation of genes involved in inflammatory responses (e.g., Tnfrsf9, Il1b, Ifitm1, Ccl17) and upregulation of genes involved in phagocytosis (e.g., Trem2, Axl, Mertk, Cd68). 2 FC>0.5, downregulation: log 2 FC<-0.5, FDR<0.05. (b) CD11c in the whole brain of 9-month-old 5XFAD mice was determined by flow cytometry + OPN + 、CD11c + OPN - 、CD11c - OPN + and CD11c -OPN - The percentage of microglial subpopulations was calculated as the number of each subpopulation within the total number of brain microglia (n=3). ****p<0.0001 by one-way ANOVA with Bonferroni multiple comparison test. (c) Analysis of CD11c by immunofluorescence staining in brain cryosections of 9-month-old 5XFAD mice + OPN + 、CD11c + OPN - and CD11c - OPN - Fractions of microglial subpopulations. Cortex / hippocampus (C / H): The percentage of each microglial subpopulation was calculated as its number in the C / H region to its total number in the whole brain (90 fields of view from 3 mice were analyzed). Within the C / H region, the percentage of each microglial subpopulation in the peri-plaque region (within 25 μm of the Aβ plaque core) was calculated as the number of each microglial subpopulation located in the peri-plaque region to its total number in the whole brain (33 fields of view from 3 mice were analyzed). The percentage of microglia distal to the plaque was calculated as the number of each microglial subpopulation located >25 μm of the Aβ plaque core to its total number in the whole brain (33 fields of view from 3 mice were analyzed). Within the peri-plaque region, the Aβ uptake cells of each microglial subpopulation were calculated as the number of Aβ uptake cells that uptake Aβ + The number of microglia in each microglia subset present in the whole brain (33 ROIs from 3 mice were analyzed). One-way ANOVA with Bonferroni multiple comparison test, ***p<0.001, ns: not significant. (d) Pie chart summarizing the number of CD11c in the brain of 9-month-old 5XFAD mice. + OPN + 、CD11c + OPN - and CD11c - OPN - The pie charts are representative results of 3 independent experiments. (e, f) CD11c in 9-month-old 5XFAD mice + OPN + and CD11c + OPN - Representative flow cytometry plots and quantification of TNF-α and TREM2 expression in microglia. + OPN + Microglia produce large amounts of TNF-α but do not express CD11c + OPN -Compared with the control group, the expression level of TREM2 was decreased (n=3). Two-tailed Student's t-test, ****p<0.0001, **p<0.01. (g) CD11c in 9-month-old 5XFAD mice + TNF-α + CD11c + TNF-α - Flow cytometric analysis of TREM2 expression in microglia. + TNF-α - Compared with microglia, TREM2 expression is higher in CD11c + TNF-α + Significantly lower in microglia (n=3). Two-tailed Student's t-test, ***p<0.001. (h) CD11c in 9-month-old 5XFAD mice + OPN + 、CD11c + OPN - and CD11c - OPN - Flow cytometric analysis of αVβ3 expression in microglial subsets. CD11c - OPN - Microglia do not express large amounts of αVβ3, but CD11c + OPN + and CD11c + OPN - Microglial subsets expressed comparable levels of αVβ3 (n=3). One-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, ns: not significant. (i) CD11c uptake of Aβ in brain cryosections of 9-month-old 5XFAD and OPN-KO.5XFAD mice + Quantification of microglia. Aβ uptake was calculated as the uptake of Aβ + CD11c + Microglia CD11c around plaques + Number within total microglia (24 ROIs from 3 mice per group analyzed). ****p<0.0001 by two-tailed Student's t-test. (j) CD11c in 9-month-old 5XFAD and OPN-KO.5XFAD mice + Flow cytometric analysis of microglial TNF-α production.CD11c in OPN-KO.5XFAD mice compared with age-matched 5XFAD +Microglial TNF-α expression was significantly reduced (n=3). ***p<0.001 by two-tailed Student's t-test. (k) Flow cytometry analysis showing CD11c from 9-month-old 5XFAD and OPN-KO.5XFAD mice. + αVβ3 + CD11c + αVβ3 - TNF-α production in microglia. OPN deficiency in 5XFAD mice leads to CD11c + αVβ3 + Significant reduction in TNF-α in microglia, but not CD11c + αVβ3 - TNF-α was not reduced in microglia (n=3). One-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, ns: not significant. (1) CD11c in 9-month-old 5XFAD mice and OPN-KO.5XFAD mice + Flow cytometric analysis of TREM2 expression in microglia. OPN deficiency results in CD11c in 5XFAD mice + The expression of TREM2 in microglia was significantly increased (n=3). **p<0.01 by two-tailed Student's t-test.

[0037] Figures 15A to 15B show immunofluorescence staining of CD11c microglia 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 antibody or tyramide signal amplification (TSA) reagent were used as negative controls. Scale bar = 50 μm. (b) CD11c in the brain of 9-month-old 5XFAD mice + 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 + , yellow arrows). Scale bar = 25 μm.

[0038] Figures 16A to 16E show microglial expression of TREM2, TNF-α, and classical OPN receptors. (a) CD11c in 9-month-old 5XFAD mice + OPN + and CD11c + OPN - Representative flow cytometry plots and quantification of TREM2 mean fluorescence intensity (MFI) in microglial subsets. + OPN + Compared with the subpopulations, CD11c + OPN - Microglial subpopulations show increased TREM2 + The percentage of cells was 74.3%, while TREM2 MFI was comparable between these two microglia subsets (n=3). Statistical analysis was performed by two-tailed Student's t-test. (b) CD11c in 9-month-old 5XFAD mice - OPN - Representative flow cytometry plots of TNF-α and TREM2 expression in microglial subsets. CD11c - OPN - Microglia express low levels of TNF-α and negligible levels of TREM2. The figure is a representative result from 3 independent experiments. (c) Microglial expression of classical OPN receptors in 5XFAD mice at different stages of the disease. The expression of αVβ3 gradually increased (n=3), while the expression of CD44 or αVβ5 was almost undetectable in microglia of 5XFAD mice during disease progression. The histogram of CD44 and the contour plots of αVβ3 and αVβ5 are representative results from 3 independent experiments. (d) OPN-αVβ3 interaction on CD11c + Effects of TNF-α production on microglia CD11c in 9-month-old OPN-KO.5XFAD mice + In microglia, upregulated TNF-α production induced by rmOPN was completely abolished by αVβ3 inhibitor (n=3). ****p<0.0001 by one-way ANOVA with Bonferroni multiple comparison test. (e) CD11c in 9-month-old 5XFAD and OPN-KO.5XFAD mice + Representative flow cytometry plots and quantification of TREM2 MFI in microglia (n=3). OPN-KO.5XFAD mice showed TREM2 + Microglia on CD11c + The percentage of microglia increased, while TREM2 MFI was comparable between the two groups. Two-tailed Student's t-test was performed for statistical analysis.

[0039] Fig.17 Figure 2 shows the fractionation of microglial subpopulations in the brain of 5XFAD mice. The schematic diagram shows that <30% of the CD11c - OPN - Microglia were enriched in the cortex / hippocampus (C / H) region of the 5XFAD mouse brain, and only 13% were located in the peri-plaque region. They were less capable of Aβ uptake (approximately 2%) and expressed marginal levels of TNF-α or TREM2. Most CD11c + OPN - Microglia (about 70%) are located in C / H areas. They are highly enriched in the periplaque area (63%) and may be protective because most of them in the periplaque area (about 60%) can take up Aβ, but produce negligible levels of TNF-α while expressing high levels of TREM2. In contrast, CD11c + OPN + Microglia subpopulations, which were also significantly enriched in the peri-plaque region (~60%), showed low levels of Aβ uptake (~7%), but almost 60% of microglia produced TNF-α and expressed low levels of TREM2, supporting the CD11c + OPN + Microglia may represent a pathogenic microglial subpopulation.

[0040] Figures 18A to 18D show in vitro analysis of OPN-dependent inhibition of the TREM2-lysosomal engulfment pathway. (a) Scheme of in vitro analysis of OPN-dependent inhibition of the TREM2-lysosomal engulfment pathway in 9-month-old 5XFAD and OPN-KO.5XFAD mice. (b) CD11c in OPN-KO.5XFAD mice compared to 5XFAD mice + TREM2 expression was significantly higher in microglia, and its expression was sharply reduced in response to rmOPN. Neutralizing anti-OPN Ab abolished the effect of rmOPN (n=3). Two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, ***p<0.001, **p<0.01. (c) CD11c in OPN-KO.5XFAD mice compared with 5XFAD mice + CD68 expression was higher in microglia, and its expression was significantly reduced by rmOPN, while anti-OPN Ab completely abolished the effect of rmOPN (n=3). ****p<0.0001, ***p<0.001 by one-way ANOVA with Bonferroni multiple comparison test. (d) CD11c + Microglia (CD11c+ CD68 + ) in lysosomes after 1 hour of incubation with FAM-Aβ 1-42 The MFI of Aβ was defined as 1h . Determination of FAM-Aβ 1-42 CD11c 24 hours after discontinuation + Microglia (CD11c + CD68 + ) and defined it as the MFI of FAM-Aβ retained in the lysosomes of 24h Compared with 5XFAD mice, CD11c + Microglia showed a higher rate of Aβ degradation [(AβMFI 1h -Aβ MFI 24h ) / AβMFI 1h ], while rmOPN significantly reduced the rate of Aβ degradation. Anti-OPN Ab completely blocked the effect of rmOPN (n=3). **p<0.01, *p<0.05 by one-way ANOVA with Bonferroni multiple comparison test. All data are expressed as mean±sem.

[0041] Figures 19A to 19J show that OPN impairs Aβ plaque compaction by inhibiting the TREM2-lysosomal engulfment pathway. (a, b) CD11c in brain cryosections of 9-month-old 5XFAD and OPN-KO.5XFAD mice + TREM2 + Representative immunofluorescence images and quantification of microglia. CD11c + TREM2 + The proportion of microglia in OPN-KO.5XFAD mice was twice that in 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) CD11c located within 25 μm of Aβ plaques in 9-month-old 5XFAD and OPN-KO.5XFAD mice + Microglia (defined as plaque-associated CD11c + CD68 in microglia + Representative immunofluorescence images and quantification of the area of ​​CD11c in 5XFAD mice due to OPN deficiency + CD68 in microglia +Increase in area (n=20 ROIs from 3 mice / group). *p<0.05 by two-tailed Student's t-test. Scale bar=25 μm. (e, f) Plaque-associated CD11c in 9-month-old 5XFAD and OPN-KO.5XFAD mice + Cathepsin B in microglia + Representative immunofluorescence images and quantification of the area of ​​CD11c in OPN-KO.5XFAD compared to 5XFAD. + Cathepsin B + (g) Representative immunofluorescence images of brains from 9-month-old 5XFAD mice and OPN-KO.5XFAD mice stained with 6E10 (red) and Thioflavin-S (green), showing two different forms of Aβ plaques. Total plaques were defined as 6E10 (red) and thioflavin-S (green). + White arrows indicate diffuse plaques (6E10 + Thio-S - ) and compacted plaque (6E10 + Thio-S + ) is indicated by yellow arrows. Scale bar = 100 μm. (h, i) Total area of ​​each plaque in 3-month-old, 6-month-old, and 9-month-old 5XFAD mice and OPN-KO.5XFAD mice (6E10 + ) and compaction area (6E10 + Thio-S + ) (n = 30 to 40 compacted plaques from 3 mice / group). Two-way ANOVA with Bonferroni multiple comparison test, ****p < 0.0001, *p < 0.05, ns: not significant. (j) Compactness index of each compacted plaque in 3-month-old, 6-month-old, and 9-month-old 5XFAD mice and OPN-KO.5XFAD mice. The compactness index was calculated as 6E10 + Thio-S + Area / 6E10 + Area (n=30 to 40 compacted 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 presented as mean±sem.

[0042] Figures 20A to 20I show that increased microglial OPN production is positively correlated with AD severity in human AD brain. (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, while 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 multiple comparison test. (b) Brain OPN expression levels were positively correlated with CDR scores in AD patients (CDR≥1, n=11), MCI patients (CDR 0.5, n=10) and controls (CDR 0, n=11). By Pearson correlation analysis, r = 0.5046, p = 0.0032. (c, d) Representative immunofluorescence images of the middle frontal gyrus from AD patients and normal controls stained for Iba-1 (microglia, red), CD11c (green), and OPN (cyan) indicated by white arrows. CD11c in the brains of AD patients (CDR ≥ 1, n = 8) was significantly higher than that of MCI (CDR 0.5, n = 9) and control subjects (CDR 0, n = 5). + OPN + The percentage of microglia was significantly higher. Each point represents 10 to 12 fields of view in each brain sample. + OPN + Microglia relative to total Iba-1 + Mean percentage of cells. Scale bar = 25 μm. One-way ANOVA with Bonferroni multiple comparison test, ****p < 0.0001, ns: not significant. (e) CD11c in brain + OPN + The percentage of microglia was positively correlated with the CDR score in AD patients (CDR ≥ 1, n = 8), MCI patients (CDR 0.5, n = 9), and controls (CDR 0, n = 5). By Pearson correlation analysis, r = 0.8383, p < 0.0001. (f, g) Brain OPN concentration and CD11c by Pearson correlation analysis + OPN + The percentage of microglia was positively correlated with the neuritic plaque score (r = 0.4919, p = 0.0043; r = 0.8226, p < 0.0001, respectively). (h, i) Pearson correlation analysis showed that brain OPN levels and CD11c + OPN +The percentage of microglia was positively correlated with the tangle assessment (r=0.4884, p=0.0046; r=0.7434, p<0.0001, respectively). All data are expressed as mean±sem.

[0043] Fig.21 Shown is the brain penetration of an unmodified KK-Ahx-Angiopep2-conjugate compared to a modified KK-Ahx-Angiopep2-conjugate in one embodiment.

[0044] Fig. 22 An exemplary Angiopep-2 conjugated anti-OPN / anti-CD11b mAb in one embodiment is shown.

[0045] Fig.23 An exemplary model depicting how OPN may function in Alzheimer's disease is shown. OPN's inhibition of microglial Aβ plaque compaction and promotion of inflammatory responses leads to cognitive impairment.

[0046] Figure 24A to Figure 24C show exemplary data associating OPN expression with disease progression in the 5XFAD mouse model. As shown, (A) microglia are the main cell source for producing OPN in the brain of 5XFAD mice during disease progression (n=3). In (B) and (C), during disease progression, compared with age-matched wild-type mice, microglial OPN expression in mice increased at both mRNA and protein levels (n=3-4). By two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, ***p<0.001, *p<0.05. These figures are respectively the same as Figure 11A, Figure 11B and Figure 11C.

[0047] Figures 25A to 25B show exemplary data of OPN expression in CD11c+ microglia in the 5XFAD mouse model. As shown, (A) during 5XFAD disease progression, OPN production was limited to CD11c+ (n=3). ****p<0.0001. (B) During disease progression, the proportion of CD11c+OPN+ microglia in 5XFAD mice increased compared to age-matched wild-type mice (n=3). By two-way ANOVA with Bonferroni multiple comparison test, ****p<0.0001, **p<0.01. These figures are the same as Figures 11D and 11E, respectively.

[0048] Figures 26A to 26F show exemplary data of OPN deficiency in reducing inflammatory response, plaque area and diffuseness, neurological dystrophy and cognitive impairment in the 5XFAD mouse model. As shown, (A) OPN deficiency reduces microglial TNF-α production (n=3, *WT vs. 5XFAD, # 5XFAD and OPN - / - .5XFAD, ****p<0.0001, ##### p<0.0001). (B) and (C) OPN deficiency leads to a decrease in the area of ​​Aβ plaques in 5XFAD mice, as well as (D) diffuse plaques (6E 10+Thio-S - ) decreased in percentage of total plaques (6E10+) (n=3). (E) OPN deficiency reduced the number of dystrophic neurites in 5XFAD mice (n=3). (F) Genetic deletion of OPN rescued cognitive impairment in 5XFAD mice (n=3-5, # :WT and 5XFAD, # p<0.05, ## p<0.01, #### p<0.001, #### p<0.0001; *: 5XFAD and OPN - / - .5XFAD, **p<0.01). Analyzed by two-way ANOVA with Bonferroni multiple comparison test. These figures are the same as FIG. 11F , FIG. 11G , FIG. 11H , FIG. 11I , FIG. 11K , and FIG. 11I , respectively.

[0049] Figures 27A to 27E show promotion of pro-inflammatory and suppression of CD11c in 5XFAD mice + Exemplary data of OPN in the phagocytic response of microglia. (A) OPN deficiency in CD11c+ microglia leads to downregulation of inflammatory response-related genes and upregulation of phagocytosis-related genes in 9-month-old 5XFAD mice, as shown in the heat map. (log 2 FC>1 or log2FC<-1, FDR<0.05). (B) Compared with 5XFAD with disease progression, OPN - / - .5CD11c expressing TNF-α in XFAD mice + The number of microglia was significantly reduced (n=3). **** p<0.0001, ***p<0.001. (C) In 5XFAD mice, OPN deficiency led to a significant decrease in TNF-α expression in CD11c+αVβ3+ microglia, but not in CD11c+αVβ3- microglia (n=3). Two-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001, *p<0.05. (D), (E) In situ analysis of brain cryosections showed that OPN deficiency in 5XFAD mice led to a significant decrease in TNF-α expression in CD11c+αVβ3+ microglia, but not in CD11c+αVβ3- microglia (n=3). Two-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001, *p<0.05. - / - Compared with 5XFAD mice, Aβ phagocytosis by CD11c+ microglia was reduced in 9-month-old 5XFAD mice. Yellow arrows indicate Aβ material that has been phagocytosed inside microglia. Aβ phagocytosis rate was calculated as the percentage of Aβ phagocytosis by CD11c+ microglia. + Aβ phagocytosis by microglia + Area (20 CD11c+ microglia from 3 mice). Two-tailed Student's t-test, *** p<0.001. Fig. 27A is the same as Fig. 14A.

[0050] Figures 28A to 28E show exemplary data of OPN inhibiting the TREM2-Axl-lysosomal pathway in CD11c+ microglia from 5XFAD mice. As shown, (A) TREM2 expression in CD11c+ microglia and CD11c- microglia was analyzed by FACS in 5XFAD mice during disease progression. TREM2 is only expressed by CD11c+ microglia (n=3). ****p<0.0001, *p<0.05. (B) (C) CO11c+TREM2+ microglia in OPN - / - .5XFAD mice were twice as high as those in 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 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 significant increase in cathepsin B+ area was observed in CD11c+ microglia in the cortex and hippocampus of OPN- / -.5XFAD compared to 5XFAD (cortex: 22 plaques from 3 mice, hippocampus: 11 plaques from 3 mice). By two-tailed Student's t-test, ****p<0.0001, ′**p<0.01. Figure 28B is the same as Figure 19A. Fig. 28C is the same as Fig. 19B. Fig. 28D is the same as Fig. 19D. Fig. 28E is the same as Fig. 19E.

[0051] Figures 29A to 29D show exemplary data of OPN inhibiting compaction of Aβ plaques by CD11c+ microglia in 5XFAD mice. (A) 9-month-old 5XFAD mice and OPN stained with 6E10 (red) and Thioflavin-S (green) - / - .5XFAD mouse brain showing two different forms of Aβ plaques. White arrows indicate diffuse plaques (6E10+Thio-S - ) and compacted plaques (6E10+Thio-S - ) indicated by yellow arrows. (B) (C) 5XFAD and OPN at 3, 6, and 9 months of age - / - . Total area (6E10+) and compacted area (6E10+Thio-S per plaque in 5XFAD mice + ) (30 to 40 compacted plaques from 3 mice per group). (D) 5XFAD mice and OPN at 3, 6, and 9 months of age. - / - .Compactness index of each compacted plaque in 5XFAD mice. The compactness index was calculated as 6E10+Thio-S + Area / 6E10 + Area (30 to 40 compacted plaques from 3 mice per group). ****p<0.0001, **p<0.01 by two-way ANOVA with Bonferroni's multiple comparison test. Figures 29A to 29D are the same as Figures 19G to 19J, respectively.

[0052] Figures 30A to 30B show exemplary data correlating brain OPN levels with Alzheimer's disease severity in the human brain. (A) OPN protein expression in homogenates of the middle frontal gyrus 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 is increased in AD patients compared to normal controls. **p<0.01 by one-way ANOVA with Tukey's multiple comparison test. (B) Brain OPN expression levels are positively correlated with CDR scores in AD patients. MCI patients and controls. By Pearson correlation analysis, r=0.5046, p=0.0032. Figure 30A is the same as Figure 20A. Figure 30B is the same as Figure 20B.

[0053] Figures 31A to 31C show exemplary data correlating the number of CD11c+OPN+ microglia with the severity of Alzheimer's disease in the human brain. (A) The middle frontal gyrus of AD patients and controls was stained for Iba-1 (microglia, red), CD11c (green), and OPN (cyan). (B) CD11c in the brain of AD patients (CDR≥1, n=8) was significantly higher than that of MCI (CDR 0.5, n=9) and control subjects (CDR 0, n=5). + OPN + The percentage of microglia increased significantly. Each point represents 10 to 12 fields of view in each brain sample. + OPN + Microglia relative to total Iba-1 + Average percentage of cells. HPF: high power field. One-way ANOVA with Tukey's multiple comparison test, ****p<0.0001. (C) CD11c in brain + OPN + The percentage of microglia was positively correlated with the CDR score in AD patients, MCI and controls. By Pearson correlation analysis, r = 0.8383, p < 0.0001. Figures 31A to 31C are the same as Figures 20C to 20E, respectively.

[0054] Figures 32A-32B show exemplary data correlating brain OPN levels and the number of CD11c+OPN+ microglia with plaque scores in human brains from Alzheimer's disease patients. (A) (B) Pearson correlation analysis of brain OPN expression (r=0.4919, p=0.0043) and CD11c + OPN + The percentage of microglia (r=0.8226, p<0.0001) was positively correlated with plaque assessment. Figure 32A is the same as Figure 20F. Figure 32B is the same as Figure 20G.

[0055] Figures 33A-33B show exemplary schematics (A) and data (B) for the generation and binding activity of Angiopep-2 conjugated monoclonal antibodies. (A) Generation of AF488-labeled Angiopep-2 α-CD11b mAb conjugate (K: lysine). (B) After incubation with microglia from 5XFAD mice (n=3), Angiopep-2 conjugated and unconjugated α-CD11b mAbs showed similar binding activity (AF488CD11b + microglia) and mean fluorescence intensity (MFI).

[0056] Fig.34Exemplary data for Angiopep-2-conjugated anti-CD11B monoclonal antibody and brain penetration of the antibody in 5XFAD mice are shown. Angiopep-2-conjugated α-CD11b mAb showed increased brain penetration in the brains of 5XFAD mice (n=3) (AF488 + microglia), as detected by surface fluorescent CD11b + As judged by the number of microglia, ****p<0.0001 by two-tailed Student's t-test.

[0057] Fig.35 Exemplary data for an anti-OPN monoclonal antibody that inhibits microglial TNF-α production in microglia from 5XFAD mice are shown. In vitro assay: 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 (MPIIIB10(1)) for 24 hours, followed by flow cytometry analysis of TNF-α production. Microglia incubated with an isotype control (mouse IgG1) were used as negative controls.

[0058] Fig.36 An exemplary schematic diagram of OPN-dependent regulation of Aβ breakdown and compaction by microglia is shown.

[0059] Fig.37 An exemplary schematic diagram of the mechanism of action of OPN is shown. A pathogenic CD11c+ microglia subpopulation producing OPN that promotes the development of AD was identified. + OPN + Contribution of microglia to AD pathology and cognitive impairment. Development of therapeutics that target OPN and improve disease pathology and cognition in preclinical AD models.

[0060] Fig.38 Exemplary data showing the effect of genetic deletion of OPN in 5XFAD mice on Aβ breakdown and plaque processing.

[0061] Fig.39 Exemplary data related to OPN in Alzheimer's disease in 5XFAD mice and humans are shown.

[0062] Fig.40 Schematic diagrams and data are shown for embodiments of the therapeutic compounds disclosed herein.

[0063] Fig.41 Exemplary schematic diagrams of embodiments of therapeutic compounds disclosed herein are shown.

[0064] Figures 42A to 42G show that the administration of anti-OPN mAb inhibits microglial proinflammatory response and improves Aβ plaque pathology. (A) Schematic diagram of anti-OPN mAb administration. Anti-OPN mAb or isotype control (mouse IgG1) was injected intravenously (10 mg / kg) into 5XFAD mice weekly for 1 or 2 months starting at 6 months of age. Microglial proinflammatory response and Aβ plaque pathology were analyzed after 1 or 2 months of treatment. (B) The percentage of CD11c+ microglia and TNF-α+CD11c+ microglia was determined by flow cytometry. Compared with age-matched 5XFAD mice (n=3) treated with isotype-matched IgG1, treatment with anti-OPN mAb for one month resulted in a moderate reduction in CD11c+ microglia and a 35% reduction in TNF-α expression of CD11c+ microglia. 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). **p<0.01, *p<0.05, ns: not significant by two-tailed Student's t-test. (C, D) Representative immunofluorescence images of brains from 5XFAD mice after 1 or 2 months of treatment with anti-OPN mAb or isotype control. Immunofluorescence is shown after staining with 6E10 (red) and Thioflavin-S (green) to distinguish between condensed and diffuse forms of Aβ plaques. Scale bar = 100 μm. (EG) Quantification of total plaque area (6E10+), diffuse plaque area (6E10+Thio-S-), and compactness index of plaques in 5XFAD mice after 1 or 2 months of treatment with anti-OPN mAb compared to isotype-treated mice (n=3). The compactness index was calculated as 6E10+Thio-S+area / 6E10+area (n=9 fields from 3 mice / group). *p<0.05, ns: not significant by two-tailed Student's t-test. All data are presented as mean±sem.

[0065] Figures 43A to 43C show that the administration of cyclic RGD (Cilengitide) inhibits the pro-inflammatory response of microglia. (A) Schematic diagram of the administration of cyclic RGD (Cilengitide). Cilengitide or vehicle control (saline) was injected intravenously (25 mg / kg) or delivered intranasally (10 mg / kg) into 5XFAD mice weekly for 1 or 2 months starting at 6 months of age. The pro-inflammatory response of microglia was analyzed after 1 or 2 months of treatment. (B) Intranasal (IN) delivery of Cilengitide for 1 month resulted in a decrease in CD11c in 5XFAD mice compared to control mice (n=3) treated with vehicle (saline). + Microglia tend to decrease and CD11c +In 5XFAD mice (n=3), IV injection of cilengitide for 1 month reduced the expression of CD11c + The percentage of microglia was reduced by 50% and CD11c + TNF-α production by microglia was reduced by approximately 50%. *p<0.5. (C) Intranasal (IN) delivery of cilengitide for 2 months reduced the percentage of CD11c+ microglia by 35% and resulted in approximately 45% reduction in TNF-α expression by CD11c+ microglia. In 5XFAD mice, IV injection of cilengitide for 2 months reduced the percentage of CD11c+ microglia by 60% and reduced CD11c+ microglia TNF-α production by 55% compared to vehicle (saline) treated control mice (n=3). *p<0.05, **p<0.01, ***p<0.001.

[0066] Figures 44A to 44C show intranasal delivery of anti-CD11b monoclonal antibodies (e.g., antibodies without conjugated -KK-amino acid bridge-AhX linker-Angiopep-2 portions). Antibodies (10 mg / kg body weight) were delivered intranasally to 6-month-old 5XFAD mice (n=4) at time 0. The antibodies were labeled with the fluorophore AF488. Observations were obtained 3 hours after administration. The control was an intravenous injection of the same non-conjugated anti-CD11b monoclonal antibody. (A) Anti-CD11b monoclonal antibody fluorescence (green) in the brain administered intranasally compared to control intravenous administration is shown. (B) Anti-CD11b monoclonal antibody fluorescence (green) in the brain administered intranasally is shown, and immunofluorescence (red) of anti-Iba-1 antibodies is also shown. Iba-1 is a microglia marker. The combined immunofluorescence (yellow) confirms the binding specificity of anti-CD11b monoclonal antibodies to microglia. (C) shows data from these studies calculating the penetration of anti-CD11b mAbs into the brain, which was calculated as the number of fluorescent microglia per microscopic field (μm) for anti-CD11b mAbs administered via the intranasal route compared to intravenous administration. 2 ×10 6 ). The data indicated that intranasal administration of the antibody resulted in approximately 10-fold increase in brain penetration compared to intravenous injection.

[0067] Figure 45A to Figure 45C show example results related to OPN and inflammasome activation. (A) shows the activation of caspase-1 and its specificity. Intracellular caspase-1 activity was measured by bioluminescence assay of microglia from 9-month-old 5XFAD mice. The specific detection of caspase-1 activity was confirmed by selective caspase-1 inhibitor (Ac-YVAD-CHO, 1 μM). The bar graph is a representative result from three independent experiments. (BC) shows the effect of OPN-αVβ3 interaction on microglial caspase-1 activation (B) and IL-1β production (C). After providing αVβ3 inhibitor to microglia of 9-month-old 5XFAD and OPN-KO.5XFAD mice (n=3), the activation of caspase-1 and IL1β by rmOPN was blocked. One-way ANOVA with Bonferroni's multiple comparison test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns: not significant. UD was not detected. DETAILED DESCRIPTION

[0068] Microglia are a type of macrophage found in the central nervous system (CNS). This article discloses a subpopulation of microglia that is CD11c+ and produces osteopontin (OPN) in the brain. CD11c+ microglia can contribute to neuronal synaptic elimination by engulfing synaptic proteins in early development, and mediate proinflammatory responses during aging, and this activity is inhibited when OPN is absent. CD11c+OPN- microglia engulf Aβ (about 60%), express high levels of TREM2, produce negligible levels of TNF-α, and can be protective. In contrast, only a small portion of CD11c+OPN+ microglia ingest Aβ, these cells express low levels of TREM2, and many produce TNF-α. The OPN production of these cells can reflect the enhanced proinflammatory response in activated lysosomes and the impaired TREM2-dependent Aβ plaque consolidation. CD11c+OPN+ microglia can represent a pathogenic microglia subpopulation and may cause symptoms of Alzheimer's disease (AD). These cells could be targets for therapeutic approaches.

[0069] The transport of substances (e.g., therapeutic agents) from blood to brain can be regulated by capillary endothelial cells in the brain, known as the blood-brain barrier (BBB). The transport of substances through the BBB is restrictive and selective. Molecules with certain properties can pass through the BBB by passive diffusion or active / facilitated transport and transcytosis. However, many substances are excluded from transport through the BBB. In some cases, various strategies can be used to make molecules that do not usually pass through the BBB pass through the BBB.

[0070] Disclosed herein are methods for transporting compounds across the blood-brain barrier (BBB). The compound may include a payload or an active agent, including a therapeutic agent, a cytotoxic agent, an imaging agent, etc. The compound typically includes a portion for delivering the payload or active agent across the BBB. In some embodiments, the active agent may be conjugated to the portion. In some embodiments, the payload or active agent may be a therapeutic agent for treating brain disease or pain. In some embodiments, the therapeutic agent may be an antibody or protein-based therapeutic agent.

[0071] In some embodiments, the portion for delivering the payload / active agent across the BBB may include a carrier agent. The portion may include a bridge, which may be an amino acid bridge (e.g., arginine, histidine, and / or lysine residues) that may be positively charged. The portion may include a joint, which may be a peptide joint, which may be a flexible joint. The portion may include one or more of a carrier agent, a bridge, a joint, and a combination thereof.

[0072] In some embodiments, brain diseases or afflictions can be treated by 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.

[0073] In some embodiments, compounds and methods for transporting therapeutic agents across the BBB can be used to target microglia with OPN (e.g., CD11c+OPN+microglia). In some embodiments, the therapeutic agent can be an antibody specific for CD11c, OPN, or CD11c and OPN. These antibodies can target CD11c+OPN+microglia. In some embodiments, the therapeutic agent can be an antibody specific for OPN.

[0074] Aspects of the invention relate to engineered brain penetrating therapeutic compounds. Aspects of the invention also relate to methods for treating neurological disorders such as Alzheimer's disease and dementia.

[0075] The specific implementation of one or more preferred embodiments is provided herein. However, it should be 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 the basis of the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in any appropriate manner.

[0076] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. In the claims and / or specification, when used in conjunction with the term "comprising," the use of the word "a" or "an" may mean "one," but is also consistent with "one or more," "at least one," and "one or more than one."

[0077] Wherever the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they should be understood to be followed by the phrase "and but not limited to." Similarly, "example," "exemplary," etc. should be understood to be non-limiting.

[0078] The term "substantially" allows deviations from the descriptor that do not adversely affect the intended purpose. Even if the term "substantially" is not explicitly recited, the descriptive term should be understood to be modified by the term "substantially".

[0079] The terms "comprising" and "including" and "having" and "involving" (and similarly "comprises," "includes," "has," and "involves") and the like are used interchangeably and have the same meaning. Specifically, each of these terms is defined to be consistent with the common U.S. Patent law definition of "comprising," and thus should be interpreted as open terms meaning "at least the following," and should also be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means a process that includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it should be understood to mean "one or more," unless such an interpretation does not make sense in the context.

[0080] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the numerical values ​​set forth. Typically, the term "about" is used herein to modify the numerical values ​​above and below the stated value by a variation of up or down (higher or lower), such as 20%.

[0081] Aspects of the invention relate to methods of treating a neurological disease. For example, the method comprises administering to a subject a therapeutically effective amount of a composition as described herein. In another example, the neurological disease may include a neurodegenerative disease. In another example, the neurodegenerative disease may include Alzheimer's disease.

[0082] As used herein, "treatment" may refer to the management and care of a subject for the purpose of combating a condition, disease or disorder in any manner that improves or otherwise beneficially alters one or more symptoms of a disease or disorder. The term "treatment" may also refer to both therapeutic treatment and prophylactic or preventive measures, where the purpose is to prevent or slow down (mitigate) undesirable physiological changes or disorders. The term may include a full range of treatments for a particular condition from which a patient suffers, such as the administration of an active compound for the following purposes: to alleviate or relieve symptoms or complications; to delay the progression of a condition, disease or disorder; to cure or eliminate a condition, disease or disorder; and / or to prevent a condition, disease or disorder, where "prevention" may refer to the management and care of a patient for the purpose of preventing the development of a condition, disease or disorder, and includes the administration of an active compound to prevent or reduce the risk of the onset of symptoms or complications. "Treatment" may also refer to prolonging survival compared to the 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 predisposed to suffering from a condition or disorder, or those in which a condition or disorder is to be prevented. The subject patient to be treated may be a mammal, such as a human. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use for treating a disease provided herein.

[0083] In an embodiment, a method of preventing or treating a neuropathy in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a composition as described herein.

[0084] The term "neuropathy" refers to any disorder of the central or peripheral nervous system of a mammal. The term "neuropathy" includes neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis), neuropsychiatric diseases (e.g., schizophrenia and anxiety disorders, such as generalized anxiety disorder). Exemplary neuropathies include MLS (cerebellar ataxia), Huntington's disease, Down syndrome, multi-infarct dementia, status epilepticus, contusions (e.g., spinal cord injury and head injury), viral infection-induced neurodegeneration (e.g., AIDS, encephalopathy), epilepsy, benign amnesia, closed head injury, sleep disorders, depression (e.g., bipolar disorder), dementia, movement disorders, psychosis, alcoholism, post-traumatic stress disorder, etc. "Neuropathy" also includes any disorder associated with the disorder. For example, methods for treating neurodegenerative disorders include methods for treating memory loss and / or cognitive loss associated with neurodegenerative disorders. Exemplary methods will also include treating or preventing neurodegenerative disorders that are unique to neurodegenerative disorders. "Neuropathy" also includes any disease or disorder (eg, cardiovascular disease) that involves, at least in part, a monoamine (eg, norepinephrine) signaling pathway.

[0085] As used herein, "Alzheimer's disease," "Alzheimer's disease," or "AD" is a disease in which cognitive function is gradually impaired over time, and includes a symptomatic pre-dementia stage manifested as mild cognitive impairment (MCI), and a dementia stage in which there is significant impairment in social or occupational functioning.

[0086] As used herein, "diagnosis" or "prognosis" refers to the use of information (e.g., genetic information or data from other molecular tests, biological or chemical information from biological samples, signs and symptoms, physical examination results, cognitive performance results, etc.) to infer the most likely outcome, time frame and / or response to a given treatment for a given disease, disorder or condition based on comparison of multiple individuals sharing common nucleotide sequences, symptoms, signs, family history or other data relevant to the patient's health status in question, or confirmation of whether a subject has mild cognitive impairment (MCI) (e.g., Alzheimer's type cognitive impairment).

[0087] As used interchangeably herein, "subject," "individual," or "patient" may refer to a vertebrate, such as a mammal, e.g., a human. Mammals may include, but are not limited to, rodents, monkeys, humans, farm animals, sport animals, and pets. The term "pet" 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.

[0088] The term "administering" may refer to introducing a pharmaceutical composition or formulation as described herein into a subject. One route of administration of the composition is intravenous administration. However, any route of administration may be used, such as oral, topical, subcutaneous, peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal, intravenous, introduction into the cerebrospinal fluid, or instillation into a body compartment. While not wishing to be bound by theory, in some embodiments, administering an active agent that includes a moiety for delivering the active agent across the blood-brain barrier may result in the active agent crossing the blood-brain barrier and entering the brain.

[0089] In some embodiments, nasal or intranasal administration can be used to administer and deliver the active agent to the central nervous system (e.g., brain). In some embodiments, the nasal or intranasal delivery of active agents can use liquid nasal sprays. Although not wishing to be bound by theory, the intranasal administration of active agents can cause the olfactory transfer of active agents to the brain. In some embodiments, the nerve cells of the olfactory epithelium present in the nasal cavity and protruding into the olfactory bulb of the brain can provide a connection between the brain and the external environment. In some embodiments, the active agent administered intranasally can move 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 portion for delivering the active agent across the blood-brain barrier.

[0090] In some embodiments, intranasal administration may result in the active agent entering the bloodstream. The active agent may then cross the blood-brain barrier and enter the brain. In some embodiments, these active agents may include one or more parts that facilitate delivery to the brain. The active agent may enter the brain via different mechanisms.

[0091] The term "therapeutically effective amount" may refer to an amount of a compound or pharmaceutical composition administered that will alleviate to some extent one or more symptoms of the disease or condition being treated and / or will prevent, or will prevent to some extent, one or more symptoms of the condition or disease that the subject being treated suffers from or is at risk of suffering from. In one embodiment, a therapeutically effective amount may refer to an amount required to treat a neurological disease (such as Alzheimer's disease) or at least one pathological effect caused by the presence of a neurological disease in a subject human or animal.

[0092] As used herein, "active agent" or "payload" refers to a substance such as a therapeutic agent, cytotoxic agent, imaging agent, etc., which is part of the compounds disclosed herein and is transported across the blood-brain barrier (BBB). "Therapeutic agent" refers to a substance that, when administered to a subject, can treat a disease or condition in the subject.

[0093] As used herein, "adsorption-mediated transcytosis" or "AMT" refers to the adsorption of substances at the luminal surface of endothelial cells, and the exocytosis of substances at the abluminal surface of endothelial cells. Typically, this transport across endothelial cells involves vesicles.

[0094] As used herein, "antibody" refers to one or more molecules that bind to an antigen. As used herein, "antibody" refers to all types of antibodies, fragments and / or derivatives. Antibodies include polyclonal antibodies and monoclonal antibodies of any suitable isotype or isotype subclass. As used herein, antibodies may refer to, but are not limited to, Fab, F(ab')2, Fab' single-chain antibodies, Fv, single chains, monospecific antibodies, bispecific antibodies, trispecific antibodies, multivalent antibodies, chimeric antibodies, canine-human chimeric antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibodies transplanted with CDRs, shark antibodies, nanobodies (e.g., antibodies composed of a single monomer variable domain), camelid antibodies (e.g., from the camelid family), microbodies, intracellular antibodies (e.g., intracellular antibodies) and / or defucosylated antibodies and / or their derivatives. Antibody mimetics are also provided. In some embodiments, the antibodies disclosed herein are active agents as part of the compounds disclosed herein that can cross the blood-brain barrier.

[0095] As used herein, "blood-brain barrier" or "BBB" refers to the cellular barrier between the blood and substances in the blood and the brain. Typically, the BBB is composed of endothelial cells and other cells.

[0096] As used herein, "bridge" may refer to a portion of a compound or moiety disclosed herein. A bridge may connect other components in a compound or moiety. In some embodiments, the 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 a compound or moiety (e.g., a therapeutic agent, a carrier agent, a flexible linker, etc.). In embodiments, the bridge may cross the blood-brain barrier. In embodiments, the bridge may cross the blood-brain barrier by adsorption-mediated transcytosis (AMT).

[0097] As used herein, "carrier agent" may refer to a first substance that facilitates the passage of a second substance across the blood-brain barrier, wherein the second substance does not normally pass through the BBB alone. A carrier agent is a portion of a compound or moiety disclosed herein. In an embodiment, a carrier agent can pass through the blood-brain barrier. In an embodiment, a carrier agent can pass through the blood-brain barrier via specific interactions with corresponding receptors expressed on cells of the blood-brain barrier. In an embodiment, a carrier agent passes through the blood-brain barrier via receptor-dependent transcytosis (RDT).

[0098] As used herein, "compound" refers to one or more active agents linked to one or more moieties. Typically, active agents and moieties (e.g., carrier agents, bridges, linkers, and combinations thereof) comprise the compounds described herein. In some embodiments, the active agent may be conjugated to the moiety. When the active agent is conjugated to a moiety, the "compound" may include the active agent, the moiety, and the conjugate.

[0099] As used herein, "conjugated to" may refer to the chemical linking of one substance to another. In this context, a payload or active agent is typically conjugated to a moiety that crosses the blood-brain barrier. "Conjugate" may refer to a chemical link connecting an active agent and a moiety.

[0100] As used herein, a "linker" is a part of certain embodiments of the compounds or moieties disclosed herein. In some embodiments, a "flexible linker" refers to amino acids that, when added to a protein or polypeptide compound or moiety, can increase the flexibility of the protein or polypeptide.

[0101] In this article, "part" refers to one or more substances that promote the active agent to cross the BBB that are connected to the active agent. In various embodiments, as described in this application, the part can include a carrier agent, a carrier agent plus a bridge, a carrier agent plus a linker, or a carrier agent plus a bridge and a linker.

[0102] As used herein, "microglia" refers to a population of macrophages from the central nervous system (CNS). In the brain, these cells can remove damaged and infected cells.

[0103] Herein, "osteopontin" refers to a secreted phosphoprotein encoded by the SPP1 gene of humans and the Spp1 gene of mice.

[0104] As used herein, "receptor-dependent transcytosis" or "RDT" may refer to the binding of a substance to a receptor on the luminal surface of an endothelial cell and the exocytosis of the substance at the luminal surface. Typically, this transport across endothelial cells involves vesicles.

[0105] As used herein, "5XFAD mouse" refers to a mouse model that recapitulates the amyloid pathology of Alzheimer's disease (Oakley, Holly et al. "Intraneuronal β-amyloid aggregates, neurode generation. 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).

[0106] Osteopontin (OPN) Antibody

[0107] Unique recombinant monoclonal OPN antibodies are described herein. "Recombinant" as it relates to polypeptides (such as antibodies) or polynucleotides refers to forms of polypeptides or polynucleotides that do not occur naturally, a non-limiting example of which can be formed by combining polynucleotides or polypeptides that do not normally exist together. As used herein, "polypeptide" can encompass the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other terms used to refer to one or more chains of two or more amino acids may be referred to herein as "polypeptides", and the term "polypeptide" may be used in place of or interchangeably with any of these terms. "Polypeptide" may also refer to the product of post-expression modification of the polypeptide (including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage or modification by non-naturally occurring amino acids through known protecting / blocking groups). The polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. With regard to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions or additions to nucleic acid sequences, peptide sequences, polypeptide sequences or protein sequences that change, 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 change causes an amino acid to be substituted by 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 may exhibit increased cross-reactivity to OPN compared to unmodified OPN antibodies.

[0108] For example, a "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include 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), non-polar 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 by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced by a structurally similar string that differs in the order and / or composition of the side chain family members.

[0109] The OPN antibodies described herein bind to osteopontin. In one embodiment, the OPN antibody has high affinity and high specificity for osteopontin. In some embodiments, the osteopontin is the 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 may 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 / sab42000 1 8), clone 53 (https: / / www.enzolifesciences.com / ADI-905-629 / osteopontin-monoclonal-antibody-53 / ), etc. Other anti-osteopontin antibodies may be used.

[0110] Some embodiments are also characterized by antibodies having a specified percentage identity or similarity to the amino acid sequence or nucleotide sequence of the anti-OPN antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, and each sequence can be compared for the purpose of comparison. When the position in the compared sequence 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, when compared to a specified region or full length of any of the anti-OPN antibodies described herein, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity. For example, when compared to a specified region or full length of any of the anti-OPN antibodies described herein, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity. Sequence identity or similarity to nucleic acids and proteins of the present invention can be determined by sequence comparison and / or contrast by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al., ed. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual comparisons or visual inspection can be used to determine the percentage sequence identity or similarity to nucleic acids and proteins of the present invention.

[0111] Aspects of the invention provide isolated antibodies specific for OPN. As used herein, the term "isolated" with respect to cells, nucleic acids (such as DNA or RNA) refers to molecules that are separated from other DNA or RNA present in the natural source of the macromolecule, respectively. The term "isolated" may also refer to nucleic acids or peptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, "isolated nucleic acid" may include nucleic acid fragments that do not exist naturally as fragments and are not found in the natural state. "Isolated" may also refer to cells or polypeptides separated from other cellular proteins or tissues. Isolated polypeptides may include both purified polypeptides and recombinant polypeptides.

[0112] As used herein, "antibody" or "antigen-binding polypeptide" may refer to a polypeptide or polypeptide complex that specifically recognizes an antigen and binds to the antigen. The antibody may be a complete antibody and any antigen-binding fragment or single chain thereof. For example, "antibody" may include any protein or peptide containing such a molecule, which comprises at least a portion of an immunoglobulin molecule having a biological activity of binding to an antigen. Non-limiting examples are the complementary determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" may refer to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen binding site that specifically binds to an antigen (immunoreacts with an antigen). The so-called "specifically binds" or "immunoreacts with..." means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides.

[0113] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as an F (ab')2 、F (ab)2 、F ab ′、F ab , Fv, scFv, etc. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" may include aptamers (such as spiegelmers), miniantibodies, and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The 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', and F(ab') 2 , Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAbs (domain antibodies), miniantibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0114] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V L) fusion protein of the variable region of the single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed by a gene fusion comprising a VH encoding gene and a VL encoding gene connected by a peptide encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, and can be rich in serine or threonine for solubility, and the VH and VL regions can be connected. H The N-terminus of L The C-terminus of the antibody is connected to the C-terminus of the antibody, or vice versa. The protein retains the specificity of the original immunoglobulin, despite the removal of the constant region and the introduction of a linker. Many methods have been described to identify the chemical structure for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules, which will fold into a three-dimensional structure that is 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.

[0115] Antibody molecules obtained from humans fall into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chains 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 some subclasses among them (e.g., γ1 to γ4). Certain classes also have subclasses, such as IgG 1 IgG 2 IgG 3 and IgG 4 etc. Immunoglobulin subclass (isotype) (e.g. IgG 1 IgG 2 IgG 3 IgG 4 IgG 5The immunoglobulin 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 molecules.

[0116] Light chain is classified as kappa or lambda (κ, λ). Every kind of heavy chain classification can be combined with kappa or lambda light chain. Generally speaking, light chain and heavy chain are covalently bound to each other, and when immunoglobulin is generated by hybridoma, B cell or genetically engineered host cell, the "tail" part of two heavy chains is bound to each other by covalent disulfide bond or non-covalent bond. In heavy chain, amino acid sequence extends from the N-terminal at the forked end of Y configuration to the C-terminal at the bottom of each chain.

[0117] Both light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used in terms of functional significance. The variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The term "antigen binding site" or "binding portion" may refer to the portion of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") region of the heavy chain ("H") and the light chain ("L") . The three highly different segments in the V region of the heavy and light chains, called "hypervariable regions", are inserted between the more conservative flanking segments called "framework regions" or "FR". Therefore, the term "FR" may refer to the amino acid sequence that naturally occurs between the hypervariable regions of immunoglobulins and adjacent to the hypervariable regions. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of the heavy chain and the light chain are each called "complementarity determining region" or "CDR".

[0118] The six CDRs present in each antigen binding domain are short non-continuous amino acid sequences that are specifically positioned to form an antigen binding domain because the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids in the amino acids in the antigen binding domain (FR region) show less intermolecular variability. The framework region mainly adopts a β-sheet conformation, and the CDR forms a loop connecting the β-sheet structure (and forming a part of the β-sheet structure in some cases). The framework region is used to form a scaffold that provides a CDR with the correct orientation through interchain non-covalent interactions. The antigen binding domain formed by the positioned CDR provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. For the heavy chain variable region or the light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDR and framework regions, respectively, as they have been previously defined (see "Sequences of Proteins of Immunological Interest," Kabat, E. et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J Mol. Biol., 196:901-917 (1987)).

[0119] In the case where there are two or more definitions of a term used and / or accepted in the art, the definition of the term as used herein is intended to include all such meanings, unless there is a clear statement to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites present in the variable regions of heavy chain polypeptides and light chain polypeptides. Such specific regions have been described by Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J Mol. Biol. 196: 901-917 (1987), the entire contents of which are incorporated herein by reference. The definition of CDR according to Kabat and Chothia includes overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition of CDRs for referring to antibodies or variants thereof is intended to be within the scope of the terms as defined and used herein. The appropriate amino acid residues covering the CDRs defined by each reference in the references cited above are listed in the following table for comparison. The exact number of residues encompassing a given CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of the antibody, one skilled in the art can determine in a routine manner which residues comprise a given CDR.

[0120] CDR Kabat Number Chothia Number VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0121] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. A skilled person can clearly assign the "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0122] In addition to the above table, the Kabat numbering system also describes the following CDR regions: CDR-H 1 starts at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5 to 7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at the fifteenth residue after the end of CDR-H 1, includes about 16 to 19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at about the thirty-third amino acid residue after the end of CDR-H2; includes 3 to 25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 starts at about residue 24 (i.e., after the cysteine ​​residue); includes about 10 to 17 residues; and ends at the next tryptophan residue. CDR-L2 starts at about the sixteenth residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins at about the thirty-third residue after the end of CDR-L2 (ie, after the cysteine ​​residue); includes about 7 to 11 residues and ends at the sequence F or WGXG, where X is any amino acid.

[0123] As used herein, the terms "nanobody" and "isolated VHH domain" are used interchangeably and refer to camelid single domain antibody fragments. "Nanobody" refers to the smallest antigen-binding fragment or single variable domain ("VHH") derived from naturally occurring heavy chain antibodies. Nanobodies are derived from heavy chain-only antibodies and are found in camelids. In the "camelid" family, immunoglobulins lacking light polypeptide chains are found. "Camelids" include Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., Lama paccos, Lama glama, Lama guanicoe and Lamavicugna). Nanobodies with low specificity bind to multiple different epitopes (or polypeptide regions) via a single antigen binding site or binding domain, whereas Nanobodies with high specificity bind to one or several epitopes (or polypeptide regions) via a single antigen binding site or binding domain.

[0124] It should be noted that the term "Nanobody" as used herein in its broadest sense is not limited to a specific biological source nor to a specific method of preparation. For example, the Nanobodies of the invention can generally be obtained as follows: (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 (e.g. from a mammalian species, such as from a human), or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques to prepare 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) any combination of one or more of the above.

[0125] As used herein, the term "monomer" refers to an antigen-binding molecule with a heavy chain variable domain without a light chain variable domain. The monomer can bind to the antigen in the absence of a light chain and has three CDR regions, named CDRH1, CDRH2 and CDRH3. A heavy chain IgG monomer has two heavy chain antigen-binding molecules connected by a disulfide bond. The heavy chain variable domain comprises one or more CDR regions, such as CDRH3 region. "VhH" or "VHH" refers to the variable domains of a heavy chain antibody (such as a single antibody). "Camelid monomer" or "Camelid VHH" refers to a monomer or an antigen-binding portion thereof obtained from a camelid source (including animals with two toes and leathery soles).

[0126] The term "DARPin" (designed ankyrin repeat protein) refers to an antibody-mimicking protein with high specificity and high binding affinity for a target protein, which is prepared via genetic engineering. DARPin is derived from natural ankyrin and has a structure comprising at least 2 ankyrin repeat motifs (e.g., comprising at least 3, 4 or 5 ankyrin repeat motifs). DARPin may have any suitable molecular weight, depending on the number of repeat motifs. DARPin includes a core portion that provides a structure and a target binding portion that is located outside the core and binds to a target. The structural core includes a conserved amino acid sequence, and the target binding portion includes an amino acid sequence that varies depending on the target. DARPin has a target specificity similar to that of an antibody. Therefore, by connecting DARPin to an antibody or an antibody fragment, such as an IgG (e.g., IgG1, IgG2, IgG3 or IgG4) antibody or a scFv-Fc antibody fragment, etc., a new form of bispecific chimeric protein is provided.

[0127] As used herein, the term "affimer" refers to a protein engineered to mimic a monoclonal antibody that binds to a target protein or peptide with high affinity, and is therefore a member of the antibody mimic family. Affibodies are composed of a three-helix bundle domain derived from the IgG binding domain of Staphylococcal protein A. The protein domain consists of a 58 amino acid sequence, 13 of which are randomized to provide a range of affibody variants. Although affibody molecules are significantly smaller than antibodies (affimers weigh about 6 kDa, while antibodies weigh about 150 kDa), affibody molecules act like antibodies because their binding sites are roughly equal in surface area to those of antibodies.

[0128] As used herein, the term "epitope" may include any protein determinant that can be specifically bound to an immunoglobulin, scFv or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, a subset of the VL domain and VH domain or complementary determining region (CDR) of an antibody is combined to form a variable region that defines a three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site present at the end of each arm of the Y. The epitope determinant can be composed of a chemically active surface grouping of a molecule (such as an amino acid or sugar side chain), and generally has a specific three-dimensional structural feature and a specific charge feature. For example, an antibody can be produced for the N-terminal or C-terminal peptide of a polypeptide. More specifically, the antigen binding site is defined by three CDRs on each of the VH chain and the VL chain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3).

[0129] In some embodiments, antibodies may 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, antibodies may be directed against human osteopontin (OPN). Osteopontin is usually secreted. There may be intracellular forms of osteopontin. For example, variants of osteopontin may be present in certain cancer cells.

[0130] In some embodiments, human osteopontin is encoded by a gene (SPP1, secreted phosphoprotein 1) having NCBI GenBank Gene ID 6696. In some embodiments, murine osteopontin is encoded by a gene (Spp1, secreted phosphoprotein 1) having NCBI GenBank Gene ID 20750. In some embodiments, osteopontin (from rat) has NCBI GenBank Reference Number: AAA41765.1 (length 317 amino acid residues), which comprises the amino acid sequence of SEQ ID NO: 2:

[0131]

[0132] As used herein, the terms "immunobinding" and "immunobinding properties" may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of the immunobinding interaction can be measured using the dissociation constant (K) of the interaction. d ) indicates that K d The smaller the value, the greater the affinity. Methods well known in the art can be used to quantify the immunological binding properties of a selected polypeptide. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "binding rate constant" (K on ) and the “dissociation rate constant” (K off ) can be determined by calculating the concentration and the actual binding and dissociation rates. (See Nature 361: 186-87 (1993)). o ff / Ko n The ratio of allows the elimination of all parameters not related to affinity and is equal to the equilibrium binding constant K D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) When the equilibrium binding constant (K) is measured by a kinetic assay such as a radioligand binding assay or similar assays known to those skilled in the art such as BIAcore or Octet (B LI), the equilibrium binding constant (K) is expressed as D)≤1 μM, ≤10 μM, ≤10 nM, ≤10 pM, or ≤100 pM to about 1 pM, the antibodies of the present invention can specifically bind to the OPN epitope. For example, in some embodiments, K D K between about 1E-12M and about 1E-11M D In some embodiments, K D K between about 1E-11M and about 1E-10M D In some embodiments, K D K between about 1E-10M and about 1E-9M D In some embodiments, K D K between about 1E-9M and about 1E-8M D In some embodiments, K D K between about 1E-8M and about 1E-7M D In some embodiments, K D K between about 1E-7M and about 1E-6M D For example, in some embodiments, K D is about 1E-12M, while in other embodiments, K D is about 1E-11 M. In some embodiments, K D is about 1E-10M, while in other embodiments, K D is about 1E-9 M. In some embodiments, K D is about 1E-8M, while in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, while in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, while in other embodiments, K D In some embodiments, K D About 6E-11 M. "Specifically binds" or "has specificity for..." can refer to an antibody that binds to an epitope via its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope when the antibody binds to the epitope via its antigen binding domain more readily than the antibody would bind to a random, unrelated epitope.

[0133] For example, OPN antibodies can be monovalent or divalent and can contain single or double chains. Functionally, the binding affinity of OPN antibodies is in the range of 10- 5 M to 10-12 For example, the binding affinity of the OPN antibody is 10- 6 M to 10- 12 M, 10- 7 M to 10- 12 M, 10- 8 M to 10- 12 M, 10- 9 M to 10- 12 M, 10- 5 M to 10-11M, 10- 6 M to 10-11M, 10- 7 M to 10-11M, 10- 8 M to 10-11M, 10- 9 M to 10-11M, 10- 10 M to 10-11M, 10- 5 M to 10- 10 M, 10-M to 10- 10 M, 10- 7 M to 10- 10 M, 10- 8 M to 10- 10 M, l0- 9 M to 10- 10 M, 10- 5 M to 10- 9 M, 10- 6 M to 10- 9 M, 10- 7 M to 10- 9 M, 10- 8 M to 10- 9 M, 10- 5 M to 10- 8 M, 10- 6 M to 10- 8 M, 10- 7 M to 10- 8 M, 10- 5 M to 10- 7 M, 10- 6 M to 10- 7 M, 10- 5 M to 10-6M.

[0134] OPN protein or its derivatives, fragments, analogs, homologs or orthologs can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components (e.g., comprising the amino acid residues of SEQ ID NO: 2). OPN protein or its derivatives, fragments, analogs, homologs or orthologs coupled to proteoliposomes can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components.

[0135] Those skilled in the art will recognize that by finding out whether a human monoclonal antibody prevents the binding of a human monoclonal antibody of the present invention to OPN, it is possible to determine without undue experimentation whether the former has the same specificity as the latter. For example, if the tested human monoclonal antibody competes with the human monoclonal antibody of the present invention, as shown by a reduction in binding by the human monoclonal antibody of the present invention, then the two monoclonal antibodies may bind to the same epitope or to a closely related epitope.

[0136] Another way to determine whether a human monoclonal antibody has the specificity of the human monoclonal antibodies of the present invention is to pre-incubate the human monoclonal antibodies of the present invention with the OPN protein (with which the human monoclonal antibodies of the present invention typically react), and then add the tested human monoclonal antibody to determine whether the tested human monoclonal antibody is inhibited in its ability to bind OPN. If the tested human monoclonal antibody is inhibited, it is very likely to have the same or functionally equivalent epitope specificity as the monoclonal antibodies of the present invention. Screening for the human monoclonal antibodies of the present invention can also be performed by utilizing OPN and determining whether the test monoclonal antibody is able to neutralize OPN.

[0137] Various methods known in the art can be used to produce polyclonal or monoclonal antibodies against the protein of the present invention or against its derivatives, fragments, analogs, homologs or orthologs. (See, for example, Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference).

[0138] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immune serum. Subsequently, or alternatively, a specific antigen or epitope thereof that is the target of the immunoglobulin being explored can be fixed on a column to purify the immunospecific antibodies by immunoaffinity chromatography. For example, D. Wilkinson (The Scientist, The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28) discusses methods for the purification of immunoglobulins.

[0139] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" may refer to a population of antibody molecules containing only one molecular species of antibody molecules, which antibody molecules are composed 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 population molecules. The mAb contains an antigen binding site that can immunoreact with a given epitope of an antigen, and the given epitope of the antigen is characterized by having a unique binding affinity for the antigen binding site.

[0140] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are immunized with an immunizing agent to elicit lymphocytes that produce or can produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0141] Active Agent

[0142] Compounds disclosed herein generally include substances that are desired to be delivered to the brain, and are referred to as active agents or payloads. In some embodiments, active agents may include therapeutic agents, drugs, cytotoxic agents, imaging agents, etc. In some embodiments, active agents may include proteins. In some embodiments, active agents may include antibodies or protein-based therapeutic agents.

[0143] The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

[0144] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding 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, mitogellin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionuclides can be used to generate radioconjugated antibodies. Non-limiting examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0145] 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 dimethyl adipimidate hydrochloride), 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 toluene 2,6-diisocyanate), and bis-active fluorine-containing 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 chelator for conjugating radionucleotides to antibodies (see PCT Publication No. WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0146] Those of ordinary skill in the art will appreciate that a wide variety of moieties may be coupled 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), the entire contents of which are incorporated herein by reference).

[0147] As long as the antibody and other parts retain their respective activities, coupling can be completed by any chemical reaction that will bind the two molecules. This connection may include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. In one embodiment, the combination is covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linking agents can be used for coupling protein molecules (such as antibodies of the present invention) with other molecules. For example, representative coupling agents can include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine. This list is not intended to exhaust various types of coupling agents known in the art, but is an example of more common coupling 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. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44: 201-208 (1984) describing the use of MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)). See also, U.S. Pat. No. 5,030,719, describing the use of haloacetohydrazide derivatives coupled to antibodies via oligopeptide linkers. Non-limiting examples of useful linkers that can be used with the antibodies of the present invention include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G); (iii) SPDP, succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propionamido] hexanoate (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS (-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 2165-G) conjugated to EDC. Chem. Co., catalog number 24510).

[0148] The joints described herein comprise components with different properties, thereby producing conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Joints comprising NHS esters have lower solubility than sulfo-NHS esters. In addition, the joint SMPT comprises a sterically hindered disulfide bond and can form a conjugate with higher stability. Disulfide bonds are generally less stable than other bonds because disulfide bonds are cleaved in vitro, resulting in fewer conjugates available. For example, sulfo-NHS can improve the stability of carbodiimide coupling. When used in combination with sulfo-NHS, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis than a separate carbodiimide coupling reaction.

[0149] In some embodiments, the active agent may include antibodies or other agents targeting certain cell types associated with the disease or pain being treated (e.g., anti-osteopontin antibodies). In some embodiments, the active agent may target cancer cells or cells associated with autoimmune disorders. In some examples, the active agent may target microglia. Microglia may be CD11c+OPN+ cells. These cells may be found in, for example, people with Alzheimer's disease. In some embodiments, the active agent may be an antibody specific to osteopontin (OPN) or CD11.

[0150] In some embodiments, the active agent may include an integrin inhibitor. In some embodiments, the integrin inhibitor may include an αVβ3 inhibitor. The αVβ3 inhibitor may include, for example, antibodies such as LM609 / Avastin, CNTO95, c7E3, and 17E6. The αVβ3 inhibitor may include, for example, antagonists such as disintegratin, peptides, and non-peptide molecules (e.g., peptide mimetics, siRNA).

[0151] In some embodiments, the αVβ3 inhibitor can be a small molecule containing cyclic RGD or a derivative thereof. In some embodiments, this 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 the connection of αVβ3 and αVβ5 integrins to matrix proteins (such as vitronectin, fibronectin, fibrinogen, von Willebrand factor, osteopontin, etc.).

[0152] In some embodiments, the αVβ3 inhibitor can be administered as part of a treatment regimen 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 bridge, a linker, a carrier agent, or a combination thereof. The αVβ3 inhibitor can be unconjugated to a bridge, a linker, a carrier agent, or a combination thereof.

[0153] In the present disclosure, active agents are typically linked to one or more moieties that allow the active agent to cross the BBB. The active agent plus one or more moieties are referred to as "compounds" of the present disclosure, which are delivered across the BBB and reach the brain. However, in some embodiments, the active agent may not be linked to one or more moieties that allow the active agent to cross the BBB.

[0154] Carrier Drugs

[0155] The carrier agent can be the moiety or can be part of one or more moieties that are connected to the active agent. In some embodiments, the carrier agent can be directly connected to the active agent. In some embodiments, the carrier agent can be connected to the active agent through a conjugate. In some embodiments, the carrier agent can be connected directly or through a conjugate to a bridge or flexible linker that is connected to the active agent.

[0156] In some embodiments, the carrier agent can facilitate transport of the moiety or a compound containing the active agent and the moiety across the BBB. In some embodiments, these substances can cross the BBB via receptor-dependent transcytosis (RDT).

[0157] In some examples, a carrier agent linked to an active agent may cause the active agent to cross the BBB. In some examples, a first carrier agent by itself may not allow the active agent to cross the BBB, but may allow the active agent to cross the BBB in combination with one or more second carrier agents or other molecules (other molecules include, for example, bridges and / or flexible linkers). In some examples, a carrier agent may allow the active agent to cross the BBB at a low level, which may be increased by combining multiple identical or different carrier agents.

[0158] In some embodiments, the carrier agent can be a peptide. In some embodiments, the carrier agent as a peptide can mediate the active agent across 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). In some embodiments, the length of such a CPP can be less than 20 amino acids and can contain positively charged amino acids. CPP can pass through the cell membrane bilayer via interaction with negatively charged cell membranes.

[0159] In an embodiment, 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), DyE-γ-E-γ-EE (masking portion; SEQ ID NO: 7), GSH, HSTPSSP (SEQ ID NO: 8), DSSLFAL (SEQ ID NO: 9), etc. (Jafari, Behzad et al. "Peptide-mediated drug delivery across the blood-brainbarrier for targeting brain tumors." Expert opinion on drug delivery 16.6 (2019): 583-605.). In some embodiments, the carrier agent includes Angiopep-2. Angiopep-2 may have an amino acid sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1) or a sequence that is at least 90% identical to SEQ ID NO: 1.

[0160] In some embodiments, the peptide vector agent may include YGRKKRRQRRRPPQQ (TAT; SEQ ID NO: 10), LLIILRRRIRKQAHAHSK (pVEC; SEQ ID NO: 11), RRLSYSRRRF (SynB3; SEQ ID NO: 12), and the like.

[0161] In some embodiments, the carrier agent can be a peptide of up to 20 or 30 amino acids, which is positively charged and amphiphilic, 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.).

[0162] In some embodiments, the carrier agent can 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.).

[0163] Generally, 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.

[0164] bridge

[0165] The bridge can be the part or can be a part of one or more parts connected to the active agent. In some embodiments, the bridge can be directly connected to the active agent. In some embodiments, the bridge can be connected to the active agent through a conjugate. In some embodiments, the bridge can be directly or through a conjugate connected to a flexible linker or carrier agent, which is connected to the active agent. In some embodiments, the bridge can be positioned between the active agent and the carrier agent.

[0166] In some embodiments, the bridge can facilitate transport of the moiety or a compound containing the active agent and the moiety across the BBB. In some embodiments, these substances can cross the BBB via adsorption-mediated transcytosis (AMT).

[0167] Typically, the bridge can be designed to enhance the ability of the moiety and attached active agent to cross the BBB and be delivered to the brain. In some embodiments, the bridge can be designed to alter the charge of the compound containing the active agent (eg, active agent plus moiety).

[0168] In some embodiments, the bridge may include an amino acid in the form of a peptide or polypeptide. The bridge may include an amino acid that changes the charge of a compound containing an activating agent. In some embodiments, the amino acid constituting the bridge may be selected to increase or decrease the isoelectric point (pI) of the compound containing an activating agent. In some embodiments, the amino acid of the bridge may be selected to increase the pI of the compound containing an activating agent. In some embodiments, the amino acid of the bridge may be selected to increase the pI of the compound containing an activating agent to an alkaline level. These amino acids may be positively charged (e.g., arginine, lysine, histidine). In some embodiments, the amino acid bridge may include 2, 3, 4, 5, 6 or more continuous positively charged amino acids. In some embodiments, the bridge may include 2, 3, 4, 5, 6 or more continuous lysine residues. In some embodiments, the bridge may include 2 continuous lysine residues.

[0169] Increasing the pI of a compound can enhance the transport of the moiety or a compound containing an active agent and the moiety across the BBB. In some embodiments, this transport can occur via adsorption-mediated transcytosis (AMT). The increased positive charge of the compound can enhance the interaction between the compound and the surface of the endothelial cells that constitute the BBB. Typically, the surface of these endothelial cells may be negatively charged.

[0170] Connectors

[0171] The linker can be the part or can be a part of one or more parts connected to the active agent. In some embodiments, the linker can be directly connected to the active agent. In some embodiments, the linker can be connected to the active agent through a conjugate. In some embodiments, the linker can be directly or through a conjugate connected to a carrier agent and / or a bridge, and the carrier agent and / or the bridge is connected to the active agent. In some embodiments, the linker can be positioned between the active agent and the carrier agent.

[0172] Typically, the linker can be designed to increase the flexibility of the part and / or the compound comprising the part and the active agent. In some cases, this flexibility can promote the solvation of the part or compound. In some cases, this flexibility can reduce the aggregation of the part or compound.

[0173] In some embodiments, a linker may refer to an amino acid or peptide spacer that separates multiple domains (eg, active agent, carrier agent) within a compound (eg, protein or polypeptide) that includes an active agent and a moiety.

[0174] In some embodiments, the linker can be a flexible linker. A flexible linker can refer to a linker that can increase the flexibility of a compound or molecule when added to it, such as a protein or polypeptide. A flexible linker can be a peptide or polypeptide. Other types of peptide linkers can be rigid linkers or cleavable linkers.

[0175] In some embodiments, the flexible peptide linker may include small polar (e.g., Ser, Thr) or non-polar (e.g., Gly) amino acids. The flexible peptide linker may have a sequence of Gly and Ser residues (e.g., a "GS" linker). An example of a GS linker amino acid sequence may include (Gly-Gly-Gly-Gly-Ser) n Other types of flexible linkers may 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.).

[0176] In some embodiments, the flexible linker may include 2-aminoethoxyacetic acid (AEA), 5-aminopentanoic acid (Ava), 8-amino-3,6-dioxaoctanoic acid (PEG2 or AEEA), 12-amino-4,7,10-trioxadodecanoic acid (PEG3), and the like.

[0177] 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.).

[0178] Generally, any of these flexible linkers may be used in the compounds described herein.

[0179] Conjugate

[0180] As described herein, the moieties can be linked to the active agent using various structures. In this article, these structures can be referred to as "conjugates". Linking the active agent to the moiety can be referred to as "conjugating" or "conjugation". In some cases, when biological molecules are involved, the conjugated structure can be referred to as a bioconjugate.

[0181] In some embodiments, the conjugate can be 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, in the presence of a given enzyme, etc.

[0182] In some embodiments, conjugation or bioconjugation can be performed 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.).

[0183] In some embodiments, MFCO-N-hydroxysuccinimide ester can be used to link an active agent that is a protein (eg, an antibody) to a moiety disclosed herein.

[0184] In some embodiments, the conjugate may include sulfo-NHS ester, biotin-NHS ester, and the like.

[0185] Pharmaceutical composition

[0186] The antibodies or fragments thereof of the invention that specifically bind to the OPN protein can be administered in the form of a pharmaceutical composition to treat a neurological or neurodegenerative disease. The principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising antibodies and guidance in the selection of components are provided, for example, in the following literature: Remington: The Science And Practice Of Pharmacy 20th Edition (Alfonso R. Gennaro et al., eds.) 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.

[0187] The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the given antibody, variant or derivative thereof, the patient's age, weight, general health, sex and diet, as well as the time of administration, the rate of excretion, the drug combination and the severity of the given disease being treated. The judgment of the medical staff on such factors is within the scope of those of ordinary skill in the art. The amount also depends on the individual patient to be treated, the route of administration, the type of preparation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0188] The therapeutically effective amount of the antibody of the present invention can be the amount required to achieve the therapeutic goal. As noted herein, this can be the binding interaction between the antibody and its target antigen, in some cases, the binding interaction interferes with the function of the target. In addition, the amount to be administered will depend on the binding affinity of the antibody to its specific antigen, and will also depend on the rate at which the administered antibody is consumed from the free volume of other subjects to which it is administered. The dosage of the antigen-binding polypeptides described herein administered to a subject (e.g., a patient) is about 0.1 mg / kg patient weight to 100 mg / kg patient weight, 0.1 mg / kg patient weight to 20 mg / kg patient weight, or 1 mg / kg patient weight to 1 0 mg / kg patient weight. Due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and lower frequency administration can be used. In addition, the dosage and frequency of administration of the antibodies disclosed herein can be reduced by enhancing the intake and tissue penetration (e.g., entering the brain) of the antibodies via modification (such as, for example, lipidation). As a non-limiting example, a typical range for therapeutically effective dosing of an antibody or antibody fragment of the invention may be about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may range, for example, from twice a day to once a week.

[0189] In the case of using antibody fragments, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is useful. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. 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 may also contain more than one active compound (as required for a given indication being treated), for example, those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth inhibitor. Such molecules are appropriately combined in an amount that is effective to achieve the intended purpose.

[0190] The active ingredients can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, 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.

[0191] Preparations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0192] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles, e.g., 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 TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow release of molecules for more than 100 days, some hydrogels release proteins for shorter time periods.

[0193] The antibodies or agents of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologs can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions may include an antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in this field and is incorporated herein by reference. Non-limiting examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles (such as fixed oils) may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, the use of such media and agents in the composition is considered. Supplementary active compounds may also be incorporated into the composition.

[0194] 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 administration, for example, intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; 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 tension, such as sodium chloride or dextrose. pH can be adjusted with an acid or alkali, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0195] Pharmaceutical compositions suitable for injectable use may 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 TM (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In embodiments, the composition is sterile and is a fluid that is easy to inject. It can be stable under the conditions of manufacture and storage, and can be preserved to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained. By various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., the effect of microorganisms can be prevented. In many cases, isotonic agents can be included in the composition, for example, sugars, polyols (such as mannitol, sorbitol), sodium chloride. The extended absorption of the injectable composition can be achieved by including an agent (for example, aluminum monostearate and gelatin) that delays absorption in the composition.

[0196] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound with one or a combination of the above-listed ingredients (as required) into an appropriate solvent, followed by filtration sterilization. For example, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. For sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, producing a powder of the active ingredient plus any additional ingredients from a previously sterile-filtered solution.

[0197] Oral compositions may include an inert diluent or an edible carrier. Oral compositions may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be blended with an excipient and then used in the form of tablets, lozenges or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is orally administered, spit out or swallowed after gargling. Pharmaceutically compatible binders and / or adjuvant materials may be included in the composition as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate or orange flavoring.

[0198] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or from a nebulizer.

[0199] Systemic administration can also be carried out through mucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for permeation barriers are used in the formulation. Such penetrants are well known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream as is well known in the art.

[0200] The compounds may 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.

[0201] In one embodiment, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such preparations is obvious to those skilled in the art. These materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (liposomes comprising targeted infected cells, with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0202] Oral or parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the given therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such an active compound to treat individuals.

[0203] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0204] Other Implementations

[0205] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

[0206] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0207] Example

[0208] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes for preparing and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples for illustrative purposes only, because similar results can be obtained using alternative methods.

[0209] Example 1

[0210] Example 1 - Definition of mouse microglial subpopulations that regulate neuronal development and proinflammatory responses in the brain

[0211] summary

[0212] Expression of Itgax (encoding the CD11c surface protein) and Spp1 (encoding OPN) is associated with activated microglia that develop in the healthy brain and in some neuroinflammatory disorders. However, it is unknown whether CD11c and OPN gene expression is a consequence of microglial activation or represents part of a gene program expressed by a stable subset of microglia. Here, we show that OPN production in the brain is restricted to activation of CD11c-positive neurons following uptake of apoptotic neurons during perinatal life. - Precursor differentiation small CD11c + Our analysis showed that co-expression of OPN and CD11c genes marks a microglial subpopulation that is expressed at birth and persists into late adult life, independent of environmental activating stimuli. + Analysis of the contribution of microglial subsets to their intrinsic functions revealed that OPN is required for subset stability and for the execution of phagocytic and proinflammatory responses, in part through OPN-dependent engagement of the αVβ3 integrin receptor. + The definition of microglia into functional microglial subsets provides new insights into microglial differentiation in health and disease.

[0213] Important Statement

[0214] CD11c enriched for osteopontin gene expression + Microglia appear at different stages of brain development, aging, and several neurodegenerative disorders. Whether the co-expression of CD11c and OPN is caused by microglial activation or represents part of a subset-specific gene program is unknown. We found that this CD11c + Microglia are a subset of cells that are formed before birth when they ingest apoptotic neurons. Our analysis also shows that they represent a stable subset that requires OPN to mediate the phagocytosis of synaptic proteins, proliferate, and develop a proinflammatory phenotype. + The definition of microglia as specialized microglial subsets provides new insights into the contribution of microglial differentiation to brain development and function in health and disease.

[0215] introduce

[0216] CD11c, also known as integrin αX (encoded by Itagx), is a defining marker for dendritic cells (DCs). When paired with Itgb2, the heterodimeric receptor binds complement iC3b and mediates phagocytosis (1). A subset of CNS-resident microglia that also express CD11c develops early in life and is characteristic of microglial development in the healthy brain and in murine models of neurodegenerative diseases, including Alzheimer's disease (AD) (2-4). + Genes expressed by microglia include the Spp1 gene, which encodes osteopontin (OPN), a cytokine-like phosphoprotein that is a prominent feature of both protective and pathogenic immune responses in peripheral lymphoid tissues (4-8).

[0217] OPN is expressed as secreted (OPN-s) or intracellular (OPN-i) isoforms that are derived from a single OPN mRNA precursor following activation of immune cells (9). OPN regulation of immune responses includes the promotion of proinflammatory responses following ligation of its classical receptor, the αvβ3 integrin expressed on macrophages (10-12). OPN production by DCs also regulates the differentiation of T helper (TH) cell subsets, including TH17 cells (8), which contribute to the development of murine experimental autoimmune encephalomyelitis (EAE) (13). Recently, microglial production of OPN has been implicated in a variety of CNS pathological disorders, including multiple sclerosis (3), spinal cord injury (14), and neurodegenerative disorders, including AD and amyotrophic lateral sclerosis (ALS) (4, 15). Although CD11c + Microglia are the major source of OPN produced by activated microglia, but it is unknown whether co-expression of CD11c and OPN is a signature of microglial activation or marks a subset-specific gene program. This represents a central question in understanding microglial development, as it involves choosing between genetic mechanisms regulating subset-specific differentiation and markers of a transient activated phenotype.

[0218] Here, we report that the production of CD11c + OPN microglia represent the microglia that engulf apoptotic neurons (AN) during perinatal life after phagocytosis from CD11c - OPN - A small (<5%) subset of precursors that differentiate and represent the only microglial producers of OPN throughout life. CD11c + OPN + The subsets displayed a stable phenotype in steady state and expressed core gene programs that were independent of microglial activation. +Analysis of the contribution of OPN to microglial function showed that OPN regulates microglial proliferation and the development of a pro-inflammatory phenotype. + The definition of microglia as functional subpopulations rather than transiently activated phenotypes suggests new approaches to analyze microglial development and provides insights into the contributions of microglia to normal and dysregulated brain development.

[0219] result

[0220] CD11c + Microglia are formed during early brain development in mice when they engulf apoptotic neurons, independent of microglia. cell activation

[0221] On dendritic cells, the CD11c protein represents an essential part of the iC3b heterodimeric receptor that mediates phagocytosis (1). Microglia expressing CD11c are detectable at birth but decrease dramatically by 3 months of age (3). Therefore, we tracked CD11c expression in healthy C57BL / 6 mice from the prenatal period to adulthood. + Development of microglia. We first verified the expression of CD11c in microglia by flow cytometry. In order to distinguish microglia (CD11b + CD45 低 ) and macrophages (CD11b + CD45 高 ), we used the CCR2 marker expressed by blood-derived macrophages but not by microglia (3, 16) and the microglia-specific marker Tmem119 (17). CD11b expressing CCR2 but not Tmem119 + CD45 高 The subset was identified as macrophages, and all CD11b cells expressed Tmem119 but not CCR2. + CD45 低 Cells were confirmed to be microglia. Fluorescence minus one (FMO) negative controls and brain CD45- cells that do not express CD11c were included to confirm that CD11b + CD45 低 Specificity of CD11c staining in the microglial population (Figure 1A). We noted that CD11c + Microglia appear late in embryogenesis (E18.5), increase to approximately 7% to 8% of total microglia by postnatal day 5 (P5), and then decline to almost undetectable levels (<1%) in young adult life. + Microglia reappeared in old (6- to 9-month-old) mice and accounted for approximately 10% of total microglia ( FIG. 1B ).

[0222] Since uptake of apoptotic neurons (ANs) triggers changes in microglial gene programs, including upregulation of ItgaxRNA (encoding CD11c) (18), we asked whether CD11c - We investigated whether upregulation of CD11c expression by precursors is a direct consequence of AN engulfment during early development. We enriched CD11c microglia by negative separation using anti-CD11c-coated magnetic beads to generate CD11c microglia (purity > 99%) and incubated them with fluorescently labeled AN for 72 h, then determined the expression of CD11c - In Figure 1C, we observed that the percentage of total CD11c - 23% of microglia took up AN (AN + ), where these AN + 77.4% of microglia are CD11c + In contrast, in 77% of non-ingested ANs, CD11c - Among microglia, <1% expressed CD11c (upper panel, Figure 1C). These findings also showed that after incubation with AN, total CD11c - About 18% of microglia continued to express CD11c + , while <1.5% of microglia incubated for 72 h in the absence of AN became CD11c + (lower figure, Figure 1C).

[0223] The upregulation of CD11c expression is not simply a consequence of microglial activation, such as phagocytosis or inflammation. - Microglia were treated with different activating stimuli that mimic CNS inflammatory disorders, including LPS and amyloid β peptide (Aβ). Although these stimuli triggered significant microglial activation, as judged by increased expression of CD86 and MHCII (19), activated CD11c - Microglia did not upregulate CD11c expression (Figure 1D, Figure 1E). Together, these results suggest that CD11c is upregulated after AN ingestion during birth. + Microglial formation is independent of conventional microglial activation.

[0224] We then examined the possible mechanisms that promote CD11c +Interactions with microglia. Phagocytic receptors expressed by developing microglia that mediate uptake and clearance of apoptotic cells include TAMs (Tyro3, ​​Axl, MerTK) and the integrin αVβ3 receptor (20, 21). We analyzed the effects of specific inhibition of αVβ3 and TAM receptors on AN uptake and their effects on CD11c - Microglia acquire CD11c + The αVβ3 inhibitor cilengitide (Cil) or the pan-TAM receptor inhibitor LDC1267 (LDC) activated CD11c - The combination of these two inhibitors reduced AN uptake by microglia by 50%. - Microglial AN uptake was further reduced to background levels (Figure 1F, left panel). Consistent with the findings in Figure 1C, we noted that within 72 h, AN stimulation induced approximately 20% of the initial CD11c - Microglia expressed CD11c, whereas in the absence of AN stimulation, CD11c expression was undetectable during the same time period. Finally, inhibition of AN uptake by cilengitide or LDC1267 reduced CD11c - CD11c + The acquisition of the phenotype was reduced by approximately 50% to 75% in each case, and the addition of both inhibitors completely prevented this phenotypic shift (Figure 1F, right). These findings suggest that engulfment of apoptotic cells can trigger CD11c after AN interacts with microglial receptors including αVβ3 and TAM. - →CD11c + change.

[0225] The stability of CD11c expression on microglia is regulated by OPN

[0226] We validated microglial OPN expression by flow cytometry analysis. We used several controls, including isotype controls, and importantly, OPN-KO microglia were used as negative control cells and microglia that selectively express an intracellular isoform of OPN (OPN-i-KI) were used as positive controls. WT microglia and OPN-i-KI microglia showed similar levels of OPN staining, whereas staining of microglia from OPN-KO donors or microglia stained with isotype controls did not yield detectable signals ( Figure 2 We note that OPN production by microglia is restricted to CD11c + Microglia, and not CD11c - Microglia significantly expressed AN (Figure 3A). In addition, AN-induced CD11c - CD11c +Microglial differentiation was also accompanied by a dramatic upregulation of OPN production ( Figure 3B ).

[0227] To determine the effect of OPN on CD11c + We investigated the phenotypic effects of CD11c from OPN-KO and WT mice during early development and aging. + Microglia were characterized. Based on flow cytometry analysis, OPN deficiency resulted in a decrease in CD11c in P5, 6-month-old, and 9-month-old WT mice. + The proportion of microglia decreased by about 50% (Figure 4A). + and CD11c - In situ analysis of microglia confirmed that CD11c + The microglial properties were reduced (50% to 75% reduction). CD11c specific staining was verified using a negative control (Figure 4B).

[0228] We further defined the contribution of OPN to the stability of CD11c expression in 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 comparable (Figure 5A). OPN-deficient neonates (P5) CD11c + Microglia showed a 75% reduction in CD11c expression ( Figure 3C ), whereas OPN-deficient CD11c from 9-month-old WT mice + Microglia showed a 50% reduction in CD11c expression (Figure 3D). We examined microglial CD11c expression using organoid hippocampal slice cultures (OHSCs). + Phenotype stability in a more physiological setting. This system allows replacement of endogenous microglia following depletion of resident microglia from organotypic hippocampal tissue slices. We used CD11c + Microglia (>95% CD11c + ) to recruit microglia. WT and OPN-KO mice showed the same number of CD11c in OHSCs at day 0 + Microglia (Figure 5B). WT CD11c + Microglia retain their CD11c + Phenotype, while most OPN-deficient CD11c + Microglia were not retained ( Figure 3E,F ).

[0229] We then assessed CD11c + Microglial responses to Aβ, a pathogenic microglial stimulator that is a hallmark of age-related neurodegenerative diseases. We observed that CD11c + Microglia stably expressed CD11c after Aβ stimulation in vitro and in OHSCs, which was consistent with OPN-deficient CD11c + In contrast, microglia lost 60% to 75% of CD11c expression (Fig. 3G, 3H). Taken together, these data suggest that stable expression of CD11c by microglia may require co-expression of OPN.

[0230] CD11c + Gene profiling of microglia

[0231] We then asked about CD11c + To investigate whether microglia represent a distinct subpopulation expressing a characteristic gene profile, we used a cold isolation protocol to isolate FACS-sorted CD11c + and CD11c - Microglia were analyzed by RNA-seq and then validated using flow cytometry ( Figure 6 For example, CD11c from newborn (P5) WT mice + and CD11c - The transcriptomes of microglia were largely overlapping, sharing 10,385 genes. However, a small number of genes (12 to 15) were expressed by CD11c - uniquely expressed (12 genes), and approximately 15 genes were expressed by CD11c + Microglia uniquely expressed CD11c + and CD11c - Microglia shared 12,072 genes, while 19 genes were only expressed by CD11c + We identified CD11c in neonatal (P5) and older (9-month-old) mice. + Four genes were shared by microglia (Figure 7B), and CD11c - Microglia did not express these four genes (Figure 7C). These represent the + Genes selectively expressed by microglia that are retained from the neonatal period to adulthood in the absence of exogenous inflammatory or infectious stimuli. 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, Figure 7E). CD11c+ The expression of these proteins by microglia is independent of activation stimuli, as CD11c - Intentional activation of microglia did not induce the expression of these core proteins (CD209 and CD36) (Figure 7F). Moreover, after phagocytosis of AN, these signature genes were upregulated by CD11c - CD11c + progeny, indicating that upregulation of these genes accompanies the formation of this subpopulation during the perinatal period ( Fig. 7G ).

[0232] OPN regulates CD11c + Intrinsic functions of microglia

[0233] Microglia can help eliminate excess neuronal synapses during neonatal brain development (24). We found that CD11c + OPN + Microglia have a high phagocytic activity towards synaptosomes, and CD11c + OPN + Microglia had no such effect (Fig. 8). We asked whether OPN contributes to this microglial function. OPN is expressed as secreted (OPN-s) or intracellular (OPN-i) isoforms that are derived from different OPN translation start sites (25). We used OPN mutant mice carrying different OPN isoforms to characterize the effects of OPN isoforms on CD11c expression. + Contribution of microglial function. Complete OPN deficiency leads to CD11c + Microglial phagocytosis of synaptosomes was reduced by 45%. OPN-i was not involved in this process (Figure 8A). Similarly, in vitro analysis showed that CD11c + OPN + Microglia showed robust phagocytosis of synaptic proteins including synaptophysin and PSD95, whereas CD11c + OPN + Microglia did not show this phagocytosis. The absence of both OPN-s and OPN-i, as well as OPN-s alone, resulted in the CD11c + Microglial function was reduced by approximately 50% ( FIG. 8B ).

[0234] OPN on CD11c + Examination of the contribution of microglial proliferation revealed that selective deletion of OPN-s was sufficient to substantially reduce CD11c + We then asked whether the interaction between OPN-s and its classical receptors might contribute to this CD11c+ Microglial function. Although CD44 and αVβ3 OPN receptors (26, 27) are not expressed by microglia at any age, αVβ3 expression increases during aging ( Fig. 9 Small-molecule-mediated blockade of αVβ3 by a cyclic RGD inhibitor (cilengitide) significantly reduced OPN-dependent Ki-67 expression (Figure 8E), suggesting that CD11c + OPN-dependent proliferation of microglia reflects ligation of αVβ3 receptors. In addition, CD11c + The robust proinflammatory response of microglia was dependent on OPN-s expression ( FIG. 8F ) and reflected engagement of the αVβ3 integrin receptor ( FIG. 8G ).

[0235] This OPN-producing CD11c + The age-dependent proinflammatory phenotype of microglial subsets suggests that they may contribute to neuroinflammatory disorders. Therefore, we asked whether the production of CD11c + We investigated whether expression of genes characteristic of microglia from OPN-sufficient mice might persist in the context of chronic inflammatory stimulation that develops in 5XFAD mice, which recapitulates the amyloid pathology of Alzheimer's disease. We observed that in the presence of 5XFAD pathology, CD11c + Microglia and their CD11c - Compared with the counterparts, the representative core genes CD36 and CD209a were expressed at the protein level (Figure 10A, Figure 10B). In addition, OPN production contributes to CD11c + Microglial stability, in stark contrast, was observed in CD11c from age-matched OPN-KO.5XFAD mice + The stability of microglia in vitro and in vitro was greatly reduced (by about 40% to 80%) (Figure 10C). In the presence of Aβ, CD11c from 9-month-old 5XFAD mice + Microglia also stably expressed the CD11c phenotype, and OPN deficiency resulted in a 40% to 80% reduction in vitro and in OHSCs, respectively ( FIG. 10D ).

[0236] discuss

[0237] Microglial expression of the CD11c receptor and OPN production has been associated with microglial activation during the development of several neuroinflammatory diseases (3, 4, 7) and in response to exogenous stimuli (14). This could be a consequence of microglial activation or part of a genetic program in microglial subsets that develops at or before birth and persists into late adulthood. Our studies support the latter view that CD11c + Microglia represent a stable subpopulation that is programmed to produce OPN rather than a transiently activated phenotype.

[0238] We identified a small (<5%) proportion of OPN-producing CD11c + A subpopulation of microglia that, at birth, engulfs ANs in the absence of external activating stimuli and then detaches from CD11c - After initial formation in the perinatal period, CD11c + Microglia decline to almost undetectable levels in young adult life and then reappear in late adult life to constitute 10% to 15% of total microglia. + Microglia and CD11c in late adult life + Microglia all expressed gene signatures that were independent of cell activation.

[0239] Single-cell transcriptomics have described microglial subsets enriched for Itagax (encoding CD11c) at different ages and during the development of neurodegenerative diseases (4-7), following the description by Owens group (3), who observed that CD11c + The number of microglia peaks in the early postnatal period (P3-5) and decreases to marginal levels in young adulthood (2 to 3 months). + Analysis of microglia revealed the emergence of CD11c during late embryogenesis (E18.5) + Microglia shrink briefly and then reappear and expand into large numbers during normal aging.

[0240] Although changes in the microglial transcriptional program following phagocytosis of apoptotic cells include upregulation of Itagax gene expression (18, 28), studies of unselected microglial populations have not determined whether this reflects changes in CD11c - De novo expression of CD11c by progenitor cells or CD11c + Expansion of microglia from smaller populations. Isolation of CD11c - Analysis of the precursor response to AN showed that AN was overexpressed by neonatal CD11c -The induction of CD11c protein expression following phagocytosis of precursors was accompanied by OPN expression, suggesting that this phenotype is a direct consequence of AN-induced differentiation. This view is supported by the following findings: CD11c + Microglial formation is independent of nonspecific microglial activation, as intentional activation of CD11c by various nonspecific stimuli - Precursors failed to induce expression of the CD11c phenotype. It may be relevant that microglia in neonatal brain regions containing a large number of apoptotic cells express high levels of Itgax / CD11c and phagocytosis-related genes (3, 6, 29, 30). In addition, injection of apoptotic neurons, but not live neurons, E. coli or zymosan particles into the brain of B6 mice induces a microglial phenotype that includes upregulation of Itagax (CD11c) (18), suggesting that induction of CD11c by microglia is a tightly controlled response to apoptosis. Without wishing to be bound by theory, transformation of adult microglia later in life may be effectively promoted by other stimuli, including amyloid β alone or as a synergistic mixture of apoptotic neurons and amyloid β.

[0241] CD11c + Expression of the phenotype was dependent on OPN production, as judged by in vitro analysis and after transfer into microglia-free hippocampal tissue. + Analysis of microglial gene profiles revealed a persistent gene signature that was independent of conventional activation stimuli. - Microglia to mimic CD11c + Selective expression of these signature genes by microglia requires CD11c + and CD11c - Further transcriptome profiling and single-cell transcriptome analysis of microglia were performed to characterize in more detail the genetic landscape of this subpopulation in healthy brain development and in the face of chronic inflammatory disorders. The stable phenotype and expression of a core gene program that persists from birth to late adulthood, independent of exogenous stimuli, suggests that CD11c + Microglia may represent a microglial cell lineage. To directly address this question, more direct probing using fate-mapping techniques is needed to track CD11c in different reporter gene mouse models. + The genetic history of microglia (31, 32).

[0242] CD11c +The genetic program of microglia includes genes associated with phagocytosis and inflammation (22, 23), suggesting that CD11c + Microglia are specialized to perform these microglial functions. In fact, we noticed that CD11c + Microglia can contribute to neuronal synapse elimination by engulfing synaptic proteins during early development and mediate proinflammatory responses during aging, potentially allowing the elimination of defective or inactive synapses. + These functions of microglial subsets are regulated by OPN, as in the absence of OPN, neonatal CD11c + Microglial phagocytosis of synaptic proteins is inhibited, while CD11c + The proliferation and proinflammatory responses of microglia reflect the engagement of OPN with the αVβ3 integrin receptor. Analysis of OPN mutant mice that specifically express OPN isoforms revealed that OPN-s, but not OPN-i, is responsible for these OPN-dependent functions. + The role of microglia in promoting inflammatory responses is reminiscent of the subset of dendritic cells that express high levels of CD11c and execute OPN-dependent inflammatory responses (33). We observed that CD11c from 5XFAD mice + Microglia express signature genes at the protein level, and the OPN-dependent phenotype noted in healthy mice may allow defining their contribution to disease development.

[0243] In conclusion, we found that CD11c + Microglia (the only producers of OPN in the brain) ingest apoptotic neurons at birth and then switch from CD11c - Without wishing to be bound by theory, this OPN-producing CD11c + The microglial population represents a novel subpopulation based on its stable phenotype and expression of signature genes that are set at birth and late in adult life and that are independent of deliberate activation. + The pro-inflammatory properties of microglia suggest that these cells may contribute to the development of neuroinflammatory diseases including AD, amyotrophic lateral sclerosis, and Parkinson's disease.

[0244] Materials and methods

[0245] Mice. Wild-type C57BL / 6 (B6) and B6. Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V) 6799Vas / Mmjax (5XFAD) mice were obtained from the Jackson Laboratory. Spp1 was generated by our laboratory as previously described. flstop (OPN-KO) and Spp1 flstop Ella cre (OPN-i-KI) mice (34). flstop Mice were crossed with 5XFAD mice to breed OPN-KO.5XFAD. All mice were housed under pathogen-free conditions. All experiments were performed in accordance with federal laws and institutional guidelines approved by the Animal Care and Use Committee of Dana-Farber Cancer Institute (DFCI).

[0246] Microglia isolation

[0247] Microglia were isolated by MACS. Adult mice were anesthetized with isoflurane and perfused transcardially with ice-cold PBS, and the brain was removed and minced with a scalpel. The tissue was enzymatically dissociated using collagenase (300U / ml, Worthington) or papain (20U / ml, Worthington) and DNase I (60U / ml, Worthington), followed by a 30% Percoll gradient to remove myelin, and the precipitated cells were resuspended in MACS buffer (PBS pH 7.2, 2mM EDTA and 0.5% BSA). Total microglia were obtained by magnetic separation using CD11b microbeads (Miltenyi). In order to separate CD11c + and CD11c - For microglia, single cell suspensions were incubated with CD11c microbeads (Miltenyi), and cells magnetically bound to the column using MACS were washed extensively, and CD11c was eluted after the magnetic field was removed. + The unbound fraction was then labeled with CD11b microbeads (Miltenyi) and separated using MACS separation, and the CD11c bound to the column was eluted. - CD11b + Cells. MACS buffer was used according to the manufacturer's protocol. Unless otherwise stated, all experiments used this standard method to isolate microglia.

[0248] Microglial isolation and fluorescence-activated cell sorting for RNA-seq analysis. Single cell suspensions were prepared as previously described (6). Briefly, mice were anesthetized with isoflurane and perfused transcardially, and the brains were quickly dissected and minced on ice using a scalpel and then homogenized by Dounce approximately 20 times in ice-cold HBSS. All tools used were pre-chilled and all isolation steps were performed on ice to minimize microglial activation. The cell suspension was transferred to a pre-chilled 50 ml tube and passed through a 70 μm cell strainer, then transferred to a pre-chilled 15 ml tube and spun at 500 g for 5 minutes 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 ice-cold 40% Percoll (Sigma), diluted, and then spun at 500 g for 30 minutes. This method produces a microglial pellet at the bottom of the 15 ml tube, while Percoll and myelin are removed by vacuum aspiration. The cell pellet was washed with 10 ml of ice-cold HBSS and spun again at 500 g for 5 minutes at 4 ° C. All samples were then resuspended in ice-cold FACS buffer (0.5% BSA, 1 mM EDTA in 1x PBS) for staining. Dead cells were excluded using Ghost dye Violet 51O (1: 1000, Tonbo Biosciences). Fc receptors were blocked using CD16 / CD32 antibodies (1: 100, BD Biosciences) to avoid nonspecific staining. The single cell suspension was then stained on ice for 20 minutes with anti-CD11b (1: 100), anti-CD45 (1: 100) and anti-CD11c (1: 100) antibodies (Biolegend), and the samples were then washed with ice-cold FACS buffer and spun at 500 g for 5 minutes. The cell pellets were resuspended in 5 ml of ice-cold FACS buffer and then sorted on a BD FACSAria II using a 70 μm nozzle in purity mode at approximately 10,000 events per second. After sorting, each sample was spun down and the cell pellets were immediately stored at -80 °C until further processing.

[0249] For P5 mice, CD11c + Microglia and CD11c - Three biological replicates of microglia were sequenced. Samples were collected from 58 mice. For 9-month-old WT mice, CD11c + Microglia and CD11c - Two replicates of microglia were sequenced.Samples were collected from 44 mice (22M+22F).

[0250] Apoptotic neurons (AN): induction and labeling of CD11c- Microglial differentiation. Primary mouse neurons were prepared from B6 embryos at embryonic days 16.5 to 17.5. The brain hemispheres were isolated and released from the meninges, and the tissue was then digested with 0.25% trypsin in HBSS at 37°C for 15 minutes, followed by titration to obtain single cell preparations. The cell suspension was filtered through a 70μm cell strainer and the cells were centrifuged at 600g for 5 minutes. The cell density was determined using a hemocytometer, and the cells were plated in Neurobasal medium supplemented with 1X B27 and 500μM GlutaMax (Invitrogen). Half of the culture medium was replaced every 3 days. To induce neuronal apoptosis, primary cultured WT neurons were treated with 300μM NMDA overnight and then carefully separated from the flask by repeated washing with PBS, followed by centrifugation, and the precipitate was processed for labeling. Neurons (1×10 7 ) were carefully resuspended in 1 ml PBS and incubated in the dark for 2 hours at room temperature with 100 μg dissolved marker dye (pHrodo iFL green, Life Technologies). To block and capture residual dye, cells were diluted with PBS and harvested by centrifugation, resuspended in 1 ml FBS and washed twice with PBS. The total number of apoptotic cells was determined using Trypan Blue staining.

[0251] Flow cytometry

[0252] 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 blocking using CD16 / CD32 antibody (1:100, BD Biosciences) to avoid nonspecific staining. Appropriate microglial surface markers were used for staining, including 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-MHCII (1:100, Biolegend), 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).

[0253] Microglial OPN expression was verified by flow cytometry analysis. Microglial OPN expression was verified in 9-month-old WT mice using a conventional intracellular staining protocol. Microglia were fixed and permeabilized with intracellular fixation and permeabilization buffer (eBioscience), followed by incubation with PE-conjugated anti-OPNAb (1:10, catalog number IC808, R&D Systems) at 4°C for 30 minutes. Isotype controls (1:10, PE-conjugated goat IgG) and OPN-KO microglia were used as negative controls. Microglia that selectively express the intracellular isoform of OPN (OPN-i-KI) were used as positive controls.

[0254] Microglial CD11c expression was verified by flow cytometry analysis. Single cell suspensions from brains of 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-TMEME119 (1:200, ABCAM), and anti-CCR2 (1:100, Biolegend). CD11b was gated from single cells / live cells. + cells, and subsequently CD11b + CD45 低 Gating on microglia and CD11b + CD45 高 The gate was set to macrophages. To distinguish between microglia and macrophages, the CCR2 marker, which is expressed by blood-derived macrophages but not microglia, was used (3, 16). To further distinguish between the two cell types, the microglia-specific marker Tmem119 (17) was also included. CD11b expressing CCR2 but not Tmem119 + CD45 高 Cells were identified as macrophages, and all CD11b cells expressed Tmem119 but not CCR2. + CD45 低 Cells were confirmed as microglia. FMO negative controls were included to confirm CD11b + CD45 低 Specificity of CD11c staining in microglial populations Brain CD45- cells, which contain predominantly non-immune cells (e.g., neurons, astrocytes, oligodendrocytes) that do not express CD11c, were included as negative controls to further validate the specificity of this strategy.

[0255] Flow cytometric detection of phagocytosis of synaptic proteins by microglia. After perfusion, mouse brains were harvested and dissected, followed by removal of myelin by centrifugation on a 30% Percoll gradient. Brain pellets were stained sequentially with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and then incubated with microglial surface markers: anti-CD11b, anti-CD45, and anti-CD11c for 30 minutes. The 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) Secondary antibody (Invitrogen, 1:300) was used for staining. Samples were acquired on a CytoFLEX (Beckman Coulter) flow cytometer and subsequently analyzed using FlowJo v10 (Tree star).

[0256] Organotypic hippocampal slice cultures. Organotypic hippocampal slice cultures (OHSC) were prepared as previously described (35). Briefly, 350 μm thick hippocampal slices were prepared from newborn (P3-P5) C57BL / 6 mice and then incubated at 35°C in 5% CO2. Microglia were removed from freshly prepared slice cultures using clodronate liposomes (FormuMax), and freshly prepared OHSC were incubated with 0.5 mg / ml clodronate liposomes at 35°C for 24 hours. Subsequently, OHSC were rinsed with warm PBS before replacing the culture 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 OHSC were maintained for 7 days before the experiment. CD11c was acutely isolated from P5 or 9-month-old WT and OPN-KO mice or 9-month-old 5XFAD, OPN-KO.5XFAD mice. + Microglia. After isolation, carefully resuspend microglia in culture medium to a final concentration of 2000 cells / μl. Supplement each OHSC without microglia with 4000 cells. + Microglia reconstituted OHSC were incubated for 7 days in the presence or absence of 2 μl synthetic human Aβ peptide 1-42 (AnaSpec, 15 μM stock). Aβ treatment was repeated 4 times every other day, so each slice was treated with a total of 8 μl Aβ peptide.

[0257] Immunofluorescence staining. P5 and 9-month-old WT and OPN-KO mice were perfused through the heart with cold PBS, followed by removal of the brain and fixation in 4% paraformaldehyde (PFA) solution at 4°C overnight. The fixed brain was then rinsed with PBS and dehydrated in 30% sucrose at 4°C overnight. Brain tissue was embedded using OCT compound (Sakura Finetek), and continuous radial cryosections (10 μm) were cut using Cryostat (Leica). Brain cryosections were permeabilized for 1 hour with PBST buffer (PBS containing 0.3% Triton X-100). OHSC slices were fixed in 4% PFA solution for 30 minutes and permeabilized for 3 hours with PBST buffer. After incubation for 1 hour in a blocking solution containing 5% normal donkey serum (Jackson ImmunoResearch Lab) in PBST to prevent nonspecific binding, cryosections or OHSC sections were incubated with appropriate primary antibodies: rabbit anti-Iba-1 (1:1000, WAKO), biotin anti-mouse CD11c (N418, 1:50, Biolegend) for 24 hours. To amplify the CD11c signal, the sections were washed with PBS and incubated with biotin anti-mouse CD11c overnight, followed by incubation with HRP-conjugated streptavidin for 1 hour, and then incubated with Alexa Fluor 488-tyramide for 5 minutes to generate high-density Alexa Fluor 488 labeling of CD11c protein in situ using the Tyramide SuperBoost Kit (Invitrogen). The sections were then incubated with Alexa-594-conjugated donkey anti-rabbit IgG (1:500; Invitrogen) for 1 hour. Immunofluorescence of brain slides incubated without anti-CD11c primary antibody or tyramide signal amplification (TSA) was included as a negative control to exclude nonspecific immunofluorescence signals. DAPI (Invitrogen) was used as a nuclear counterstain (incubation for 10 min) before analyzing samples using an Olympus fluorescence microscope.

[0258] RNA profiling

[0259] 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 bioinformatics analysis (gene hit counts) were performed as follows at GENEWIZ, LLC. (South Plainfield, NJ, USA):

[0260] Library preparation. Extracted RNA samples were quantified using a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was checked using an Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). PolyA selection strategy: CD11c of P5WT was prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to the manufacturer's instructions. + and CD11c - RNA sequencing library of microglial samples. Briefly, mRNA was first enriched with oligo (dT) beads, and then the enriched mRNA was fragmented at 94 ° C for 15 minutes. The first and second strand cDNAs were then synthesized, the cDNA fragments were end-repaired and adenylated at the 3′ end, the universal adapters were connected to the cDNA fragments, and then the index was added and the library was enriched by limited cycle PCR. The sequencing library was verified on an Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA) and quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) and by quantitative PCR (KAPABiosystems, Wilmington, MA, USA).

[0261] Ultra-low input strategy: Since the number of cells obtained from adult mice is very limited, CD11c + and CD11c - Microglial samples were processed with the SMART-Seq v4 Ultra Low Input Sequencing Kit for full-length cDNA synthesis and amplification (Clontech, Mountain View, CA) and with Illumina Nextera XT libraries for sequencing library preparation. Briefly, cDNA was fragmented and adapters were added using transposase, followed by limited cycle PCR to enrich and index the cDNA fragments. The final library was evaluated using an Agilent TapeStation.

[0262] HiSeq sequencing. Sequencing libraries were clustered on the flow cell lanes prior to loading the flow cell onto an Illumina HiSeq instrument (4000 or equivalent) according to the manufacturer's instructions. Samples were sequenced using a 2×150 bp paired-end (PE) configuration, and image analysis and base calling were performed by the 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.

[0263] RNA-Seq data analysis. Mapping and gene counting were performed by GeneWiz. After checking the quality of the raw data, sequence reads were trimmed using Trimmomatic v.0.36 to remove adapter sequences and poor quality nucleotides. Trimmed reads were mapped to the Mus musculus reference genome (ENSEMBL) using STAR aligner v.2.5.2b, a splice aligner that detects and integrates 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 exonic regions were counted. Differential expression was considered significant with an FDR-adjusted p-value of <0.05.

[0264] Quantitative PCR. CD11c was extracted using the RNeasy Plus Universal Mini Kit according to the manufacturer's instructions (QIAGEN). + and CD11c - RNA from microglia. cDNA was reverse transcribed from 35 ng RNA and prepared using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. cDNA was reverse transcribed using QuantStudio TM Real-time quantitative PCR was performed on the selected core genes using the 6Flex Real-Time PCR System (Applied Biosystems). Each reaction used 5 μl of cDNA, 4.92 μl of PowerUp TM SYBR TM Green Master Mix (Applied Biosystems) and 0.08 ul of primers (IDT, working concentration: 200 nM). ΔΔ Ct method to compare gene expression levels.

[0265] In vitro synaptosome phagocytosis assay

[0266] Isolation and labeling of synaptosomes. Synaptosomes were isolated from WT mice using Syn-PER Synaptic Protein Extraction Reagent (Thermo Scientific) following the manufacturer's instructions. For pHrodo labeling, dissolved pHrodo iFLgreen (Life Technologies) was incubated with synaptosomes on a shaker in PBS at a ratio of 20 μg pHrodo / 1 mg synaptosomes for 1 hour at room temperature in the dark. Unconjugated pHrodo was removed by washing with PBS, and pHrodo-conjugated synaptosomes were then resuspended in PBS containing 5% DMSO, aliquoted and stored at -80°C until use.

[0267] Microglia phagocytosis of synaptosomes. Microglia isolated from P5 and 9-month-old WT, OPN-KO, and OPN-i-KI mice were mixed with 1×10 5 Each cell was incubated with 136 μg pHrodo Green-labeled synaptosomes for 1 hour, followed by staining with Ghost dye Violet 510 (1:1000, Tonbo Biosciences) and anti-CD11c (1:50, Biolegend) for 30 minutes. CD11c was assessed by flow cytometry analysis. + Phagocytosis of synaptosomes by microglia.

[0268] Quantification and statistical analysis. The data in the figure are expressed as mean ± sem. Statistical analysis was performed using Prism (GraphPad Prism version 9.0). Quantification of fluorescence microscopy images was performed using ImageJ. Statistical analysis was performed using GraphPad Prism by one-way or two-way ANOVA with Bonferroni multiple comparison test to compare the mean values ​​of multiple groups. Comparisons between two groups were analyzed using a two-tailed Student's t-test. P values ​​< 0.05 were considered statistically significant. All statistical details of the experiment (including the statistical tests used, the exact values ​​of sample size) can be found in the accompanying figure descriptions.

[0269] Data availability. All protocols and reagents are described in detail in the manuscript and Supporting Information. RNA-seq data have been deposited in the NCBI Gene Expression Omnibus and are available through GEO Series accession number: GSE190713.

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[0306] Example 2

[0307] Example 2 - Osteopontin-producing microglia contribute to Alzheimer's disease

[0308] Overview

[0309] Microglia are resident immune cells in the brain, and dysregulated microglial activation is a major feature of Alzheimer's disease (AD) 1Despite overwhelming evidence that this cell type plays a key role in AD, the contribution of this cell type to disease pathology is poorly understood. Evidence that microglia can play a protective role comes from the observation that disease-associated microglia (DAMs) can phagocytose amyloid-β (Aβ) plaques. 2，3 In contrast, there is evidence that microglia are pathogenic based on the observation that elimination of microglia in the 5XFAD mouse model prevents Aβ seeding and plaque formation. 4 Our studies suggest that microglial effects reflect separate contributions from protective and pathogenic microglial subsets that can be distinguished based on their developmental history and functional phenotype.

[0310] Although many disease-associated genes have been identified in mouse models of AD, most have not been confirmed in studies of human AD. The Spp1 gene (encoding OPN) is an exception, as it appears to be strongly upregulated by microglia in both AD animal models and human disease. 2,5-8 We recently characterized a microglial subset based on the expression of the CD11c surface marker that is the sole producer of osteopontin (OPN) in the healthy mouse brain during early development and adult life. 9 .

[0311] OPN is also expressed by peripheral dendritic cells and macrophages, where it can modulate inflammatory and autoimmune responses 10-12 Steinman and colleagues implicate OPN in neuroinflammatory and neurodegenerative disorders, including multiple sclerosis. 13，14 However, the contribution of OPN-producing microglia to neurodegenerative diseases is unknown.

[0312] Here we define pathogenic CD11c that drives AD pathology + OPN + Microglial subsets in 5XFAD mice are represented by CD11c + Genetic deletion of microglial OPN production inhibits inflammatory cytokine production and promotes TREM2-dependent uptake of amyloid fibrils by microglia 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 in diffuse Aβ plaques and significant improvement in cognitive function. We extended these mouse findings to human disease using clinically and neuropathologically characterized brain tissue from AD patients, mildly cognitively impaired patients, and cognitively normal controls (Mt. Sinai Brain Repository). This analysis showed that human CD11c +OPN production by microglia is closely associated with the progression of both clinical dementia and AD neuropathology. Taken together, these findings suggest that targeting CD11c + Microglial OPN production to reduce diffuse toxic plaques and inhibit neuroinflammation represents an effective AD treatment strategy.

[0313] result

[0314] OPN contributes to AD pathology and cognitive impairment in 5XFAD mice

[0315] In order to identify the cell source of OPN during disease progression in the brain of 5XFAD mice, we measured the OPN expression of astrocytes, neurons and microglia at 3, 6 and 9 months of age. Astrocytes and neurons do not produce detectable amounts of OPN, while microglia OPN production is robust and increases with disease progression (Figure 11a). Compared with the B6-WT control of age-matched, the OPNmRNA of 9-month-old 5XFAD microglia increased by 10 to 20 times and the OPN protein increased by 2 to 3 times (Figure 11b, Figure 11c), which shows that microglia OPN production is closely related to disease development.

[0316] Microglia co-expressing CD11c represent the sole producers of OPN in the healthy mouse brain 9 We observed that microglial OPN production in 5XFAD mice was also restricted to the CD11c + Microglial subsets increased significantly during the first 6 months of disease progression compared to relatively low levels in healthy age-matched controls (Fig. 11d, Fig. 11e).

[0317] We have generated 5XFAD transgenic mice with spp1 flstop OPN-KO·5XFAD mice were generated by hybridization of OPN-KO and 5XFAD mice. 15 , and OPN deficiency was confirmed at the gene and protein levels (Figure 12). Genetic deletion of OPN in 5XFAD mice resulted in a reduction in TNF-α production by microglia to levels similar to those of age-matched healthy (B6-WT) controls (Figure 1lf). Examination of Aβ plaque area in OPN-KO 5XFAD mice showed a 3- to 5-fold reduction in plaque area in both the cortex and hippocampus at 6 and 9 months of age compared to age-matched 5XFAD controls (Figure 11g, Figure 11h), suggesting that OPN may inhibit Aβ clearance in 5XFAD mice. Anti-AβmAb 6E10 (to identify diffuse and condensed forms of Aβ plaques) and Thioflavin-S (which identifies only β-sheets + Aβ condensed plaques) were used to identify Aβ plaques in the 5XFAD mice. 3) Analysis of co-stained brain cryosections also showed that genetic deletion of OPN significantly reduced the proportion of diffuse plaque development at 9 months of onset (Figure 11i).

[0318] Neuronal dystrophy is a core element of 5XFAD disease pathology and consists of swollen, bulbous neurons composed of dysfunctional axons and terminals expressing amyloid precursor protein (APP). 16 , which is associated with the clinical severity of dementia 17，18 We noticed that the number of dystrophic neurites per plaque was reduced by approximately 50% in OPN-KO.5XFAD mice (Fig. 11j, Fig. 11k). Since an increase in the number of dystrophic neurites is closely associated with cognitive decline, 19，20 We measured the effect of OPN expression on cognitive function of 9-month-old 5XFAD mice using a T-water maze to assess spatial learning and memory (acquisition test) and cognitive flexibility (reversal test). OPN deletion significantly improved these cognitive functions, as indicated by the 40% to 50% increase in correct choices made by OPN-KO.5XFAD mice in the acquisition phase and reversal phase of these tests (Figure 11l). In short, these data show that OPN deficiency significantly reduces microglial proinflammatory responses, diffuse Aβ plaques and dystrophic neurites in 5XFAD mice, and improves cognitive function.

[0319] Pathogenic CD11c in 5XFAD brain + OPN production by microglia promotes proinflammatory responses and inhibits Aβ uptake

[0320] Due to CD11c + Microglia are the only cell producers of OPN in the mouse brain (Figure 1d, Figure 11a-c), so the pathogenic effects of OPN may depend on this CD11c + Development and function of microglial subsets.

[0321] Based on purified CD11c from 9-month-old 5XFAD mice and OPN-KO.5XFAD mice + RNA-Seq analysis of microglia defines the role of OPN in CD11c in 5XFAD disease + We used flow cytometry to confirm the contribution of microglia to the phenotype of 5XFAD mice. + FACS gating of microglia ( Fig.13 OPN deficiency resulted in downregulation of inflammatory response-related genes (e.g., Tnfrsf9, il1b, Ccl1) and upregulation of phagocytosis-related genes (including Trem2, MerTk, CD68, and Ctsb) ( Figure 14a and Table 1 ), which include core components of the molecular pathways responsible for Aβ uptake and lysosomal compaction 3Table 1 includes CD11c from 9 month old OPN-KO.5XFAD mice compared to FXFAD mice. + List of differentially expressed genes (DEGs) in microglia. 2,985 DEGs were identified. If log 2 FC>1, the gene expression is considered upregulated, or if log 2 FC, -1, gene expression was considered downregulated. DEGs were considered significant with FDR-adjusted p-value < 0.05.

[0322] Flow cytometric analysis showed that the majority (80%) of microglia in the brains of 9-month-old 5XFAD mice expressed neither CD11c nor OPN (double negative, DN), whereas approximately 15% to 20% expressed both (double positive, DP), and less than 5% were CD11c + OPN-. We did not detect significant amounts of CD11c - OPN + We then determined the fraction of each microglial subpopulation within the cortex and hippocampus (C / H), the brain regions most affected by 5XFAD disease, and further evaluated the fraction of each subpopulation residing in the peri-plaque region (25 mm of the Aβ plaque core) using cryosections of brains from 9-month-old 5XFAD mice. 21 In the case of CD11c + OPN + 、CD11c + OPN - and CD11c - OPN - Prior to staining and evaluation of microglial subsets, microglial CD11c-specific immunofluorescence signals were verified using several negative controls ( FIG. 15 a , FIG. 15 b ).

[0323] About 60% CD11c + OPN + The (DP) microglia subpopulation resides in the C / H region, and almost all of these DP microglia are located in the peri-plaque region. Analysis of Aβ uptake by these peri-plaque DP microglia showed that only about 12% of microglia took up Aβ (Figure 14c, Figure 14d). A considerable portion (about 70%) of the CD11c + Subset-CD11c + OPN - - also resided in C / H regions and were mostly located in the periplaque region. However, in this case, more than 90% of the periplaque CD11c + OPN -Microglia contain ingested Aβ material. + In contrast to microglial subsets, CD11c - OPN - The majority (>70%) of the (DN) subpopulations reside in brain regions outside the C / H region. Analysis of DN subpopulations within the C / H region also showed that, although about half of the DN subpopulations were located near or within the peri-plaque region, only about 2% of the DN subpopulations contained Aβ material (Figure 14c, Figure 14d).

[0324] Since two CD11c + All microglial subsets were closely associated with Aβ plaques (Figure 14d), and we characterized their phenotypes in more detail by flow cytometry. + OPN - subpopulation (which expresses low levels of TNF-α but strongly increases TREM2 + Compared with the proportion of microglia, CD11c + OPN + (DP) showed substantial TNF-α production and reduced TREM2 expression (Figures 14e, 14f, and 16a). - OPN - (DN) microglia account for 80% of the total brain microglia, they do not produce detectable TNF-α and express marginal levels of TREM2 (Figure 16b). + Direct comparison of TREM2 and TNF-α expression in microglia showed that pro-inflammatory (i.e., TNF-α + )CD11c + Microglia contain approximately 5% TREM2 + cells, while about 15% of TNF-α - TREM2 in microglia + (Figure 14g). These analyses showed that DP CD11c + OPN + The pro-inflammatory phenotype of microglia was associated with reduced Aβ uptake (Figure 14c, Figure 14d), while CD11c + OPN - Microglia displayed robust levels of Aβ uptake.

[0325] Because genetic deletion of OPN reduced microglial TNF-α production to levels found in nondiseased mice (Fig. 1f), we examined the relationship between OPN and the proinflammatory TNF-α phenotype. OPN-dependent ligation of the macrophage αVβ3 integrin receptor promotes a proinflammatory phenotype and reduces uptake of apoptotic debris 22，23In 5XFAD mice, the classical αVβ3 integrin receptor 9 of OPN, 24 The addition of rmOPN induced a significant increase in the expression of CD11c in microglia, while other OPN receptors (CD44 and αVβ5) were almost undetectable (Figure 16c). + Microglial TNF-α expression was abolished by treatment with cyclic RGD inhibitor of αVβ3 integrin (cilengitide) (Figure 16d). We also noted that CD11c - OPN - Microglia expressed negligible amounts of αVβ3, consistent with the near absence of proinflammatory TNF-α expression by this subset, whereas CD11c + Subpopulation (CD11c + OPN - and CD11c + OPN + ) expressed higher (>30%) levels of αVβ3 (Figure 14h). + To investigate the contribution of microglia to the phenotype of microglia, we examined microglia from OPN-KO.5XFAD mice. We noted that compared with 5XFAD mice, microglia from 9-month-old OPN-KO.5XFAD mice had a CD11c + Microglia display Aβ + There was a dramatic increase in material uptake (Fig. 14i) and a 50% reduction in TNF-α production (Fig. 14j).

[0326] In conclusion, (a) OPN deficiency leads to TNF-α + Cells in αVβ3 + CD11c + Decreased proportion and TREM2 in microglia + The proportion of microglia increased and (b) lacked the OPN receptor CD11c + Microglia do not produce TNF-α (Figure 14k, Figure 14l and Figure 16e). These findings suggest that OPN can bind to CD11c through its (αVβ3) receptor. + Engagement of microglia promotes TNF-α production and reduces TREM2 expression.

[0327] Together, these findings reveal three major microglial subsets that contribute differently to 5XFAD disease. - OPN - Microglia represent the major (~80%) subset in the brain, but do not take up Aβ or produce TNF-α and are not a major component of Aβ pathology. + (OPN+ and OPN - Both CD11c glial cells make up a relatively small fraction of total brain microglia (approximately 20%), but >60% of these microglia are located within plaque areas. + OPN production by microglial subsets further complements their distinct functional phenotypes within and around plaque areas. + OPN + Microglia produced high levels of TNF-α, but relatively few contained ingested Aβ or expressed TREM2, consistent with their pathogenic contribution to the disease. + OPN - Microglia do not produce significant levels of TNF-α, but they actively phagocytose Aβ and can play a disease-protective rather than disease-driving role. (Summary in Fig.17 ).

[0328] OPN inhibits Aβ plaque compaction by suppressing the TREM2-lysosomal engulfment pathway

[0329] Recent evidence suggests that the TREM2 pathway enhances lysosomal degradation of amyloid fibrils, leading to increased extrusion of condensed Aβ at the expense of diffuse Aβ plaques in 5XFAD mice 25，26 We asked whether the reduction in diffuse plaques that we had noted earlier in OPN-KO.5XFAD brains (Fig. 11i) reflected increased activation of this TREM2-lysosomal pathway. Analysis of in situ TREM2 expression in brain cryosections confirmed flow cytometric analysis: CD11c + Microglia expressed 2- to 3-fold the TREM2 expressed in 5XFAD mice (Figure 19a, Figure 19b). This increase was associated with enhanced lysosomal activation, as determined by increased expression of CD68 lysosomal activation glycoprotein in microglia from OPN-KO.5XFAD mice compared to age-matched 5XFAD mice (Figure 19c, Figure 19d) and lysosomal expression of cathepsin B (a lysosomal cysteine ​​protease that cuts and degrades Aβ) increased 3- to 4-fold. 27，28 (Fig. 19e, Fig. 19f). Taken together, these data suggest that OPN-dependent downregulation of the TREM2-phagolysosomal Aβ compaction pathway could explain our findings that genetic deletion of OPN in 9-month-old 5XFAD mice significantly reduced diffuse plaques (6E10 + Thio-S - ) in total plaque (6E10 + ) (e.g., Figure 11i).

[0330] We then asked whether the interaction between recombinant OPN might inhibit CD11c + TREM2 expression of microglia. We found that TREM2 expression was sharply inhibited by rmOPN, and this reduction was reversed by adding anti-OPN Ab (Figure 18a, Figure 18b). In addition, rmOPN also inhibited lysosomal activation, as judged by reduced CD68 expression (Figure 18c). The inhibition of TREM2-lysosomal activation (Figures 19a to 19d and Figures 18a to 18c) was accompanied by CD11c + Aβ degradation in lysosomes of microglia [(AβMFI 1h -Aβ MFI 24h ) / AβMFI 1h ] was significantly decreased, which was completely reversed by anti-OPN Ab (Fig. 18d).

[0331] To directly test the contribution of OPN to microglial plaque compaction, we differentiated compacted from total plaques using Thioflavin-S to identify Aβ in a β-sheet conformation. + We found that, as shown in Figures 3g and 3h, the 40% to 45% reduction in total plaque area in OPN-KO.5XFAD mice was accompanied by an increase in compacted plaque area (Figure 19i), as measured by the plaque compactness index (6E10 + Thio-S + Area / 6E10 + This was judged by a significant increase in the area (Figure 19j).

[0332] Together, these data suggest that OPN-mediated inhibition of TREM2-dependent lysosomal activation, which results in impaired compaction of Aβ plaques by microglia and increased diffuse plaques, represents a potential mechanism of action of OPN in the regulation of Aβ. + CD11c + Microglia drive a pathogenic pathway that drives 5XFAD disease.

[0333] CD 11c + Increased OPN production by microglia is associated with disease severity and neurodegeneration in brain tissue from AD patients Pathologically related

[0334] To extend these findings from the mouse model of AD to human disease, we analyzed brain tissue from: clinically and neuropathologically confirmed AD patients (cohort characterization in Tables 2 and 3), the patient's clinical dementia rating (CDR) ≥ 1 at the time of death, as well as patients with mild cognitive impairment (MCI, CDR = 0.5) and cognitively normal controls (CDR = 0). Measurements of OPN in the homogenate of the middle frontal gyrus showed that OPN expression in the brains of AD patients increased 3-fold compared to cognitively normal control subjects. Increased OPN expression in the brains of AD patients was also noted compared to the brains of patients with MCI, but it 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).

[0335] We then determined CD11c by immunofluorescence analysis of human brain sections from the middle frontal gyrus, an area early affected by AD. + OPN + (DP) Percentage of microglia 29 Normal controls and MCI subjects showed similar proportions of CD11c + OPN + Microglia (CD11c + OPN + Iba-1 + We observed that CD11c in brain slices from AD patients was significantly higher than that in slices from normal controls. + OPN + Microglia increased 3-fold (Figure 20c, Figure 20d). In addition, CD11c + OPN + The percentage of microglia was significantly higher than that from MCI patients (Figure 20d), indicating that CD11c + OPN + The percentage of microglia represents a more sensitive parameter than simple OPN levels for distinguishing AD patients from patients with MCI ( FIG. 20 c ). + OPN + This conclusion was further supported by the very strong correlation between the percentage of microglia and the CDR score ( FIG. 20 e ).

[0336] We also determined the relationship between (a) OPN levels and (b) CD11c in the middle frontal gyrus with neuritic plaques. + OPN +Correlation between the percentage of microglia and the neurofibrillary tangle density assessment determined postmortem by a neuropathologist prior to storage of the samples in the Mount Sinai Brain Bank 30 We found that higher brain OPN levels were associated with higher levels of neuritic plaques (r=0.4919, p=0.0043) and neurofibrillary tangles (r=0.4884, p=0.0046) (Figure 20f, Figure 20h). Similar to the association with dementia severity, CD11c + OPN + There was a stronger correlation between the percentage of (DP) microglia and the density of neuritic plaques (r = 0.8226, p < 0.0001) and neurofibrillary tangles (r = 0.7434, p < 0.0001) (Figure 20g, Figure 20i). These findings further support the view that CD11c + OPN + The percentage of microglia can be a sensitive marker for both clinical severity and neuropathology of AD.

[0337] discuss

[0338] Microglial dysregulation characterized by sustained inflammatory responses and impaired Aβ plaque processing contributes to neuronal dystrophies and cognitive decline in the setting of AD 1，31 We have previously noted that a subset of microglia expressing CD11c, first defined by the Owens group, is the sole producer of OPN in healthy mouse brain tissue. 9 Here, we report that, during disease development, OPN production is similarly restricted to CD11c in the 5XFAD model of AD. + Here, we report that during disease development, OPN production is similarly restricted to CD11c microglia in the 5XFAD model of AD. + Microglia. The CD11c + OPN + OPN production by the subpopulations may reflect enhanced proinflammatory responses in activated lysosomes and impaired TREM2-dependent Aβ plaque consolidation. These findings provide new insights into the molecular mechanisms driving AD-related cognitive impairment and, without being bound by theory, highlight OPN as a tractable therapeutic target in AD.

[0339] Based on the expression of CD11c and OPN production, microglia can be divided into three major subsets, namely, CD11c + OPN + 、CD11c + OPN- and CD11c -OPN - Microglia. Although CD11c - OPN - The subpopulation accounts for >80% of microglia in the whole brain, <30% are resident in the cortex / hippocampus of 5XFAD mouse brains, and about 10% are located in the peri-plaque area. Without wishing to be bound by theory, since less than 2% of CD11c - OPN - Microglia take up Aβ and do not produce appreciable TNF-α, so this CD11c - OPN - Microglia represent a homeostatic subpopulation that contributes to 5XFAD pathology. In contrast, the expression of OPN is dependent on the expression of CD11c + Dissection of microglia identified a pathogenic and protective subset. + OPN - Microglia make up a small population (~3%) throughout the brain, but ~70% of this subpopulation resides in the cortex / hippocampus and the majority of these cells (~65%) are located in the peri-plaque areas. + OPN - Microglia may be protective because they robustly phagocytose Aβ (approximately 60%), express high levels of TREM2, and produce negligible levels of TNF-α. + OPN + The DP microglia subset was also significantly enriched in the peri-plaque area (approximately 60%), but only 5% to 7% of microglia took up Aβ, whereas almost 60% of microglia produced TNF-α and expressed low levels of TREM2, supporting the view that CD11c + OPN + Microglia represent a pathogenic microglial subpopulation.

[0340] TREM2 promotes Aβ uptake in microglia 32，33 , CD11c + OPN - The strong expression of TREM2 by microglia may contribute to the robust uptake of Aβ by these cells. + OPN + Weak expression of TREM2 by microglia is consistent with low levels of Aβ uptake. CD11c + OPN -Microglial uptake of Aβ may also reflect the engagement of the αVβ3 receptor by milk fat globule EGF factor 8 (MFG-E8), which binds to phosphatidylserine (PtdSer) molecules that decorate Aβ plaques and promotes microglial endocytosis. 3，34，35 Without being bound by theory, MFG-E8-αVβ3 interaction drives CD11c + OPN - Microglia take up Aβ, while competition with OPN reduces CD11c + OPN + DP microglia bind MFG-E8 and enhance pathogenic CD11c + OPN + TNF-α production by microglial subsets.

[0341] In conclusion, our findings support the notion that OPN-deficient CD11c + Microglia represent a protective subpopulation that actively takes up Aβ for lysosomal digestion without inducing an inflammatory response. In contrast and without being bound by theory, OPN-producing CD11c + These findings complement our understanding of the DAM phenotype, which is CD11c + ) understanding, which suggests that the protective contribution of DAM 2 Reflecting the CD11c + OPN - Subgroup activity.

[0342] Colonna and colleagues have demonstrated that TREM2 plays an important role in regulating the interaction between microglia and Aβ plaques, possibly by inducing microglia to surround the Aβ plaque structure and change the Aβ plaque structure to a more compact form, thereby limiting neurological damage. 36，37 Microglial Aβ compaction depends in part on a TREM2-dependent phagocytic pathway that internalizes Aβ into activated lysosomes for digestion 38 Thus, TREM2 deficiency in mouse models and AD patients leads to increased Aβ burden, diffuse plaques, dystrophic neurites, and cognitive impairment. 17，26，36，39 The contribution of TAM receptors, which may act downstream of TREM2 in this plaque processing pathway, comes from the observation that TAM receptor-deficient APP / PS1 mice display reduced numbers of dense-core plaques and increased cognitive deficits. 3OPN-dependent inhibition of Aβ phagocytosis and suppression of the TREM2-lysosomal phagocytosis pathway were confirmed by our investigation of CD11c + Transcriptomic analysis of microglia revealed OPN-dependent downregulation of key components of this pathway (Trem2, Axl, Mertk, C68, and Ctsb), as well as OPN-deficient CD11c + Microglial TREM2 expression and CD68 expression were increased. The significant reduction in plaque burden in OPN-deficient 5XFAD mice reflected accelerated lysosomal degradation of Aβ, as judged by upregulation of microglial cathepsin B and a decrease in diffuse plaques with an increase in compacted plaques.

[0343] To date, clinical trials of anti-Aβ antibodies have shown that reductions in plaque burden are not accompanied by improvements in cognitive function. Recent evidence suggests that microglial deposition of previously ingested and compacted neurotoxic Aβ fibrils via exocytosis or microglial cell death is required for the formation of non-toxic dense-core plaques that are associated with rescued cognitive function. 3，40 Without being bound by theory, microglia that accumulate Aβ fibrils into dense plaques represent a neuroprotective mechanism 3 , so antibodies targeting Aβ disaggregate cohesive, non-toxic Aβ fibrils into oligomers that increase neurotoxicity 41 Thus, without wishing to be bound by theory, indiscriminate disaggregation of dense plaques may not be effective in ameliorating neurotoxicity and associated cognitive impairment. 42，43 Without being bound by theory, since OPN deficiency increased plaque compactness, substantially reduced dystrophic neurites, and improved cognitive function, targeting OPN represents a more effective therapeutic strategy compared to the current clinical approach of indiscriminately targeting plaques in mAb trials. In contrast, OPN deficiency favorably altered the ratio of proinflammatory to phagocytic microglia and increased TREM2-associated Aβ uptake, resulting in increased conversion of neurotoxic Aβ material to nontoxic dense-core plaques.

[0344] Not all findings from AD mouse models are applicable to human AD. However, our core findings appear to be applicable to human disease. Upregulation of the OPN encoding gene (Spp1) has been identified in AD mouse models. 2，7，44 and patients 8，45，46 Interestingly, no increase in microglial Spp1 expression was noted in AD patients carrying TREM2 loss-of-function variants (TREM2-R47H and TREM2-R62H). 44Without being bound by theory, genetic impairment of the TREM2 pathway eliminates the need for OPN-dependent inhibition of the TREM2 pathway in the development of plaque-associated pathology.

[0345] Although elevated OPN levels in CSF and plasma samples have been reported in AD patients 47 , but the relationship between brain OPN levels and disease severity in AD patients has not been clearly established. Consistent with our preclinical findings in 5XFAD mice, brain OPN levels and, in particular, CD11c were expressed in postmortem brain samples from AD patients. + OPN + The proportion of microglia strongly correlated with neuritic plaque scores. Analysis of human brain samples from well-characterized AD patients and controls also provided evidence that brain OPN levels directly correlated with disease severity according to CDR scores. In addition, CD11c + OPN + The percentage of microglia increased with the progression and severity of dementia and was strongly correlated with the CDR score. CD11c was noted in MCI patients and AD patients + OPN + An increase in the percentage of microglia is a sensitive indicator of disease progression, and CD11c + OPN production by microglia is strongly correlated with both the deficits and severity of AD neuropathology. Neurofibrillary tangles, composed primarily of aggregated, hyperphosphorylated tau protein, represent another important pathological hallmark of AD. We note that brain OPN levels as well as CD11c + OPN + The percentage of microglia is closely related to neurofibrillary tangles assessment, which indicates that OPN contributes to tau pathology. These findings indicate that OPN simultaneously regulates multiple disease exacerbating factors (i.e., neuritic plaques and neurofibrillary tangles) that contribute to AD pathology. Without being bound by theory, the correlation of the results in AD mouse models and AD patients indicates that OPN is a promising therapeutic target that will be successfully translated into the clinic.

[0346] In conclusion, this report distinguishes pathogenic microglia from protective microglia through the expression of CD11c and the production of OPN. Without being bound by theory, CD11c + OPN - The microglial subset was protective because they efficiently took up Aβ without concomitant TNF-α production and expressed high levels of TREM2, while CD11c + OPN +Microglia represent a pathogenic subpopulation that produces large amounts of TNF-α and expresses low levels of TREM2. Without wishing to be bound by theory, this OPN-producing CD11c + The pathogenic effects of microglial subsets (including promotion of proinflammatory responses and inhibition of the protective TREM2-lysosomal engulfment pathway) reduce plaque dissemination and toxicity. As described above, the CD11c-producing microglial subsets first defined in the murine 5XFAD model + Microglial levels of OPN positively correlate with disease severity and neuropathology in AD patients. Therefore, targeting microglial OPN responses could be a more effective therapeutic strategy compared to the two current forms of antibody-based approaches to target amyloid plaques.

[0347] method

[0348] Mouse

[0349] C57BL / 6 (B6) and B6. Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799Vas / Mmjax (5XFAD) mice were obtained from Jackson Laboratory (MMRRC). B6. Spp1 flstop (OPN-KO) mice were previously generated by our laboratory 15 . flstop Mice were crossed with 5XFAD mice to generate OPN-KO.5XFAD mice. Age-matched and sex-balanced mice were used. All mice were housed under pathogen-free conditions. All experiments were performed in accordance with federal laws and institutional guidelines approved by the Animal Care and Use Committee of Dana-Farber Cancer Institute (DFCI).

[0350] Microglia isolation

[0351] Cell separation was performed by magnetic activated cell sorting (MACS). Adult mice were anesthetized with isoflurane and then perfused transcardially with ice-cold PBS. The brain was then minced and enzymatically dissociated using type IV collagenase (300U / ml, Worthington) or papain (20U / ml, Worthington) and DNase I (60U / ml, Worthington). Myelin was removed by a 30% Percoll gradient. The precipitated cells were then resuspended in MACS buffer (PBS pH 7.2, 2mM EDTA, and 0.5% BSA). Total microglial populations were magnetically separated using anti-mouse CD11b microbeads (Miltenyi). Unless otherwise stated, all experiments used this method to separate microglia. Astrocytes were separated using anti-mouse ACSA-2 microbeads (Miltenyi). Neurons were negatively enriched using anti-CD11b, anti-ACSA-2, and anti-O4 microbeads (Miltenyi) to deplete microglia, astrocytes, or oligodendrocytes.

[0352] Microglia isolation and fluorescence-activated cell sorting for RNA-seq analysis. Single-cell suspensions were prepared as previously described. 9，48. Briefly, mice were anesthetized with isoflurane and perfused transcardially with ice-cold PBS. The brains were quickly dissected and minced with a scalpel on ice and then homogenized by Dounce in ice-cold Hank's balanced salt solution (HBSS). The cell suspension was passed through a 70-μm cell strainer and then spun at 500g for 5 minutes at 4°C. Myelin and debris were removed using 10 ml of ice-cold 40% Percoll (Sigma) and spun at 500g for 30 minutes before washing with 10 ml of ice-cold HBSS and spun again at 500g for 5 minutes 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, TonboBiosciences) and anti-CD16 / CD32 antibodies (2.4G2, 1:100, BD Biosciences) were used to exclude dead cells and block Fc receptors, respectively. The single cell suspension was 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 on ice for 20 minutes. The samples were then washed with ice-cold FACS buffer and spun at 500g for 5 minutes. The cell pellet was resuspended in 5 ml of ice-cold FACS buffer and then sorted on a BD FACS Aria II using a 70μm nozzle in purity mode at approximately 10,000 events per second. After sorting, each sample was spun and the cell pellet was immediately stored at -80°C until further processing. For 9-month-old 5XFAD mice, CD11c + Microglia and CD11c - Two replicates of microglia were sequenced. For 9-month-old OPN-KO.5XFAD mice, CD11c + Two replicates of microglia were sequenced.

[0353] Flow cytometry

[0354] Flow cytometric analysis of microglia Ghost dye Violet 510 (1:1000, TonboBiosciences) and anti-CD16 / CD32 antibody (2.4G2, 1:100, BD Biosciences) were used to exclude dead cells and avoid nonspecific staining, respectively. Microglia were stained with appropriate surface markers including 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) and then fixed and permeabilized with an intracellular fixation and permeabilization buffer kit (eBioscience) for subsequent staining with anti-OPN (IC808, 1:10, R&D Bioscience). Intracellular staining was performed with anti-TNF-α (MP6-XT22, 1:50, Biolegend) and anti-TNF-α (Biolegend). Data were acquired on CytoFLEX LX (Beckman Coulter) and analyzed with FlowJo (Tree Star).

[0355] Validation of CD11c expression in microglia. Validation of CD11c expression in microglia was performed as previously described9. Briefly, brain single cell suspensions from 9-month-old 5XFAD mice were stained with Ghost dye Violet 510 (1:1000, TonboBiosciences) and anti-CD16 / CD32 antibodies (2.4G2, 1:100, BD Biosciences), and then incubated 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). CD11b was gated from single cells / live cells. + cells, and subsequently CD11b + CD45 低 Gating on microglia and CD11b +CD45 高 The gate was set to macrophages. Tmem119, a microglia-specific marker expressed by blood-derived macrophages but not by microglia 49 and CCR2 were included to distinguish microglia from macrophages 50，51 CD11b expressing CCR2 but not Tmem119 + CD45 高 Cells were identified as macrophages, and all CD11b cells expressed Tmem119 but not CCR2. + CD45 低 Cells were confirmed as microglia. Fluorescence minus one (FMO) negative controls were included to confirm CD11b + CD45 低 Specificity of CD11c staining in the microglial population. Brain CD45- cells, which predominantly contain non-immune cells that do not express CD11c, were included as a negative control to further verify staining specificity.

[0356] Real-time quantitative PCR (RT-qPCR). Microglial RNA was extracted using the RNeasy Plus Universal Mini Kit according to the manufacturer's instructions (QIAGEN). 100 ng of RNA was then used to reverse transcribe complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. QuantStudio TM Real-time quantitative PCR of Spp1 was performed using the 6Flex Real-Time PCR System (Applied Biosystems). Each reaction used 5 μl of cDNA, 4.92 μl of PowerUp TM SYBR TM Green Master Mix (Applied Biosystems) and 0.08 ul of primers (IDT, working concentration: 200 nM). ΔΔ Ct method was used to compare the gene expression level of Spp1.

[0357] T-shaped water maze

[0358] Animal cognitive testing was performed in the Neurobehavioral Laboratory at Harvard Medical School. The T-water maze (WTM) behavioral paradigm assesses spatial learning and memory by training mice to use spatial cues in a room to navigate to a hidden platform to escape the water. The test also measures cognitive flexibility through a reversal learning procedure in which mice must learn a new location of a hidden platform. Testing was performed as previously described. 52. The test apparatus is a cross maze made of transparent plexiglass (each arm is 14 cm long and 4.6 cm wide), and each arm is designated as north (N), south (S), east (E) or west (W). A divider is placed on the maze to block the appropriate arm so that the mouse can choose only the E arm or the W arm for escape. At the beginning of each trial, the mice are placed in the N arm or the S arm in a semi-random order. The maze is filled with water (25°C to 26°C), and the escape platform is placed on the E side of the maze, submerged about 1em below the water surface. To ensure that the mice cannot see the submerged platform, the water is made opaque by adding white, non-toxic paint. At the beginning of the test, the divider is placed in the appropriate position to block the appropriate arm and the mouse is transported to the appropriate starting point. The experimenter scores the correct or incorrect response of each trial and allows the mouse to stay on the platform for 10 seconds before being removed. The mice are tested 10 times a day, and the correct response percentage is calculated by averaging the correct responses of the 10 trials per 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.

[0359] RNA-seq analysis of CD11c microglia

[0360] RNA extraction. Following the manufacturer's instructions (QIAGEN), total RNA was extracted from FACS-sorted cell pellets using the RNeasy Plus Universal Mini Kit. Library preparation, sequencing reactions, and bioinformatics analysis (gene hit counts) were performed as follows at GENEWIZ, LLC. (South Plainfield, NJ, USA):

[0361] Library preparation. The concentration of extracted RNA was quantified using a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was determined using an Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). +The number of microglia is very limited, and microglial samples from 9-month-old 5XFAD and OPN-KO.SXFAD mice were processed with SMART-Seq v4 ultra-low input sequencing kits for full-length cDNA synthesis and amplification (Clontech, Mountain View, CA), and Illumina Nextera XT libraries were used for sequencing library preparation. Briefly, cDNA was fragmented and adapters were added using transposase, followed by limited cycle PCR to enrich cDNA fragments and add indexes to cDNA fragments. The final library was evaluated using Agilent TapeStation.

[0362] HiSeq sequencing. Sequencing libraries were clustered on flow cell lanes. After clustering, the flow cell was loaded onto 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 by HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq were converted to fastq files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.

[0363] RNA-Seq data analysis. Mapping and gene counting were performed by GENEWIZ (South Plainfield, NJ, USA). After quality checking of the raw data, sequence reads were trimmed using Trimmomatic v.0.36 to remove adapter sequences and poor quality nucleotides. The trimmed reads were mapped to the Mus musculus reference genome available at ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequence. As a result of this step, a BAM file was generated. Unique gene hit counts were calculated by using feature counts from the Subread software package v.1.5.2. Only unique reads that fell within exonic regions were counted. Significantly differentially expressed genes (DEGs) were then detected using a negative binomial model implemented in the R package edgeR 53，54 If log 2 FC>0.5, the gene expression is considered upregulated, or if log 2 If FC < -0.5, the gene expression was considered downregulated. DEGs were considered significant with a false discovery rate (FDR) adjusted p value < 0.05. FC represents the fold change per kilobase per million reads (RPKM) at FDR < 0.05.

[0364] Immunofluorescence staining

[0365] Mouse brains were removed after transcardial perfusion and fixed overnight in 4% paraformaldehyde solution (PFA, ElectronMicroscopy Sciences) at 4°C. After rinsing with PBS, the fixed brains were dehydrated in 30% sucrose at 4°C overnight. The brains were then embedded in OCT compound (Sakura Finetek) and serial radial cryosections (10 μm) were cut using a Cryostat (CM3050S, Leica).

[0366] Brain cryosections were permeabilized with PBS containing 0.1% Triton X100 (PBS-T) for 1 hour. After incubation in a blocking solution containing 5% normal donkey serum (Jackson ImmunoResearch Lab) in PBS-T for 1 hour to prevent nonspecific binding, the sections were then incubated with appropriate primary antibodies: rabbit anti-Iba-1 (1:1000, WAKO), anti-amyloid β (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), goat anti-cathepsin B (AF965, 1:100, R&D) for 24 hours. To amplify the CD11c signal, the sections were washed with PBS and incubated for 5 minutes with the Alexa Fluor488 Streptavidin Tyramide SuperBoost Kit (Invitrogen) following the manufacturer's instructions. The sections were then incubated for 1 hour 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 antibody or tyramide signal amplification (TSA) were included as negative controls to exclude nonspecific immunofluorescence signals. DAPI (Invitrogen) was used as a nuclear counterstain (incubation for 10 minutes). For Thioflavin-S staining, brain cryosections were incubated in filtered 1% Thioflavin-S aqueous solution at room temperature for 10 minutes and washed 2×3 minutes in 80% ethanol and 3 minutes in 95% ethanol before adding the blocking solution. Samples were analyzed under an Olympus fluorescence microscope. Quantification of fluorescence microscopy images was performed using ImageJ (NIH).

[0367] In vitro microglial assay

[0368] To test the OPN-αVβ3 interaction on CD11c + To investigate the effect of pro-inflammatory responses of microglia, microglia were enriched by anti-CD11b microbeads (purity > 95%, Miltenyi) from 9-month-old OPN-KO.5XFAD mice and cultured in conventional microglia culture medium (DMEM-F12 with 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / ml recombinant mouse M-CSF) at 3 × 10 5 Microglia were pre-incubated with the selective αVβ3 inhibitor cilengitide (10 μM) for 1 h, followed by the addition of 12.5 μg / ml recombinant mouse OPN (rmOPN) and cultured for 24 h, and then analyzed by flow cytometry for TNF-α CD11c + Microglial expression.

[0369] To test the effect of OPN on TREM2 lysosomal activation and Aβ degradation, microglia enriched by anti-CD11b microbeads (purity >95%, Miltenyi) from 9-month-old 5XFAD and OPN-KO.5XFAD mice were cultured at 3 × 10 cells / mL in conventional microglia culture medium (DMEM-F12 with 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / ml recombinant mouse M-CSF). 5 Cells / well were seeded into 12-well plates. Microglia were then pre-incubated with anti-OPN Ab (10 μg / ml) for 1 hour and then incubated with rmOPN (12.5 μg / ml) overnight. FAM-labeled Aβ 1-42 Peptides (1 μM) were added to the cultures for 1 hour. After 1 hour of incubation, CD11c + Microglia (CD11c + CD68 + ) in lysosomes 1-42 The mean fluorescence intensity (MFI) of Aβ was defined as 1h Then, FAM-Aβ was deactivated 1-42 The cells were cultured in the presence or absence of anti-OPN Ab (10 μg / ml) and / or rmOPN (12.5 μg / ml) for 24 h, and then analyzed by flow cytometry for the expression of TREM2, CD68, CD11c + Microglia expression and CD11c + CD68 + FAM-Aβ retained in microglia 1-42MFI (defined as AβMFI 24h The Aβ degradation rate was calculated as (AβMFI 1h -Aβ MFI 24h ) / AβMFI 1h .

[0370] Human brain sample processing and evaluation

[0371] Clinical and autopsy diagnoses, brain collection procedures, tissue processing, and assessment of AD pathology are described in detail elsewhere 26 Brain tissue of the middle frontal gyrus (an area affected early in AD) from clinically and neuropathologically characterized AD patients and controls was obtained from the Mount Sinai Brain Bank through Neurobiobank (https: / / neurobiobank.nih.gov / ) 26 Three groups of deceased persons 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 determined using the Clinical Dementia Rating (CDR) scale. 55 The CDR assesses cognitive and functional impairment 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.

[0372] Assessment of plaques and tangles in the human brain. According to the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) protocol 56 Neuropathic plaques and neurofibrillary tangles were assessed at the Mount Sinai Brain Bank. Sections from paraffin-embedded blocks were variably stained with hematoxylin and eosin, modified Bielschowski, modified Thioflavin S, and anti-β-amyloid (4G8), anti-tau (AD2). All neuropathological data regarding the extent and distribution of neuropathic lesions were collected in a blinded fashion with respect to the dementia status of the subjects. The BraakAD staging score for progression of neurofibrillary neuropathology was assigned to each case. 57，58 In addition, quantitative data on the density of neuritic plaques were collected as previously described. 30 .

[0373] Human OPN ELISA. To quantify human brain OPN concentration, 10 mg of human brain frozen tissue was homogenized in 0.3 ml lysis buffer [20 mM Tris-Hcl pH 8, 130 mM NaCl, 1% triton X100 and protease inhibitor cocktail (Roche)], kept on ice for 45 minutes and centrifuged at 13000 g for 20 minutes at 4 ° C. The protein concentration of brain lysate was measured using a BCA kit (ThermoFisherScientific), and 10 μg of protein was loaded into each well of a human OPN Quantikine ELISA plate (R&D Systems). The manufacturer's instructions were followed.

[0374] CD11c + OPN + Human brain staining of microglia. Paraffin-embedded brain tissue sections from the middle gyrus of the frontal lobe were dewaxed in xylene, rehydrated in graded ethanol, and washed with PBS (PBS-T) containing 0.1% Triton X100. Brain sections were boiled in 10mM citrate buffer (pH 6) to recover antigens and washed with PBS-T. The endogenous peroxidase activity of the sample was quenched with 3% hydrogen peroxide solution for 60 minutes, washed, and incubated with Zyblack (Zytovision, BS-0002-8) for 30 minutes to reduce autofluorescence. Endogenous biotin was blocked with a biotin blocking kit (Invitrogen, E21390). Staining was performed by multiplexing three tyramide SuperBoost kits (Invitrogen, B40936, B40912, B40923). First, the sections were blocked with blocking buffer for 60 minutes and 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)] overnight at 4°C. After washing, the sections were incubated with HRP-conjugated streptavidin for 60 minutes, washed, and incubated with Alexa Fluor TM 647 tyramide streptavidin reagent for 10 minutes, followed by incubation with the reaction stop solution for 5 minutes. TM 488tyramid anti-mouse and Alexa Fluor TM The cells were incubated with 555tyramid anti-rabbit. The nuclei were labeled with Hoechst and the slides were mounted with Immu-Mount medium (Thermofisher Scientific).

[0375] Brain slides of control (n=5), MCI (n=9), and AD (n=8) were coded and 10 to 12 images were captured in three separate sequences under a confocal microscope (Leica DMi8) under the same laser intensity conditions; 408 nm laser at 5%, 488 nm laser at 0.1%, 638 nm laser at 6.5%, and 552 nm laser at 3%, with a magnification of X400. Triple-positive cells expressing Iba-1, CD11c, and OPN were counted using ImageJ software (NIH), and total Iba-1-positive cells were counted for each section. CD11c was calculated in the total Iba-1-positive cells for each section. + OPN + Microglia (CD11c + OPN + Iba-1 + ) and the mean value was calculated for each brain sample. The analysis was performed by a blinded researcher.

[0376] Statistics and reproducibility

[0377] Data are expressed as mean ± sem. Statistical analysis was performed using GraphPad Prism software version 9.0. Analysis of multiple groups was compared by one-way or two-way ANOVA with Bonferroni test. Comparisons between two groups were analyzed using a two-tailed Student's t-test. Correlation analysis was performed using Pearson's correlation. P < 0.05 was considered significant and is represented as * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical details (including the specific statistical tests used in the different analyses, and the corresponding sample sizes) are given in the corresponding figure legends.

[0378] Data availability

[0379] CD11c + Microglia, CD11c from 9-month-old 5XFAD - Microglia and CD11c from 9-month-old OPN-KO.5XFAD mice + The RNA-seq data of microglia have been deposited in the NCBI Gene Expression Omnibus (GEO) with accession number GSE191118. The data are available upon reasonable request from the authors.

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[0416] Wang,Y.et al.TREM2-mediated early microglial response limitsdiffusion and toXicity of amyloid plaques.J Exp Med 213,667-675,doi:10.1084 / jem.20151948(2016).

[0417] Joshi.P.etal.TREM2 modulates differential deposition of modified andnon-modified Abeta species in extracellular plaques and intraneuronaldeposits.Acta Neuropathol Commun 9,168,doi:10.1186 / s40478-021-01263-x(2021).

[0418] Parhizkar,S.et al.Loss of TREM2 function increases amyloid seedingbut reduces plaque-associated ApoE.Nαt Neurosci 22,191-204,doi:10.1038 / s41593-018-0296-9(2019).

[0419] Wang,Y.et al.TREM2 lipid sensing sustains the microglial response inan Alzheimer's disease model.Cell160,1061-1071,doi:10.1016 / j.cell.2015.01.049(2015).

[0420] Baik,S.H.,Kang,S.,Son,S.M.&Mook-Jung,I.Microglia contributes toplaque growth by cell death due to uptake of amyloid beta in the brain ofAlzheimer's disease mouse model.Glia64,2274-2290,doi:10.1002 / glia.23074(2016).

[0421] Liu.Y.H.et al.An N-terminal antibody promotes the transformation ofamyloid fibrils into oligomers and enhances the neurotoxicity of amyloid-beta:the dust-raising effect.JNeuroinflammation 12,153,doi:10.1186 / s12974-015-0379-4(2015).

[0422] Panza,F.,Lozupone,M.,Logroseino,G.&Imbimbo,B.P.A critical appraisalof amyloid-beta-targeting therapies for Alzheimer disease.Nat Rev Neurol15,73-88,doi:10.1038 / s41582-018-0116-6(2019).

[0423] Long,J.M.&Holtzman,D.M.Alzheimer Disease:An Update on Pathobiologyand Treatment Strategies.Cell179,312-339,doi:10.1016 / j.cell.2019.09.001(2019).

[0424] Zhou,Y.et al.Human and mouse single-nueleus transeriptomics revealTREM2-dependent and TREM2-independent cellular responses in Alzheimer′sdisease.Nat Med 26,131-142,doi:10.1038 / s41591-019-0695-9(2020).

[0425] Grubman,A.et al.A single-cell atlas of entorhinal cortex fromindividuals with Alzheimer's disease reveals cell-type-specific geneexpression regulation.Nat Neurosci 22,2087-2097,doi:10.1038 / s41593-019-0539-4(2019).

[0426] Lau,S.F.,Cao,H.,Fu,A.K.Y.&Ip,N.Y.Single-nucleus transcriptomeanalysis reveals dysregulation of angiogenic endothelial cells andneuroprotective glia in Alzheimer's disease.Proc Natl Acad Sci U S A117,25800-25809,doi:10.1073 / pnas.2008762117(2020).

[0427] Sun,Y.et al.Elevated osteopontin levels in mild cognitive impairmentand Alzheimer's disease.Mediators of inflammation 2013,615745,doi:10.1155 / 2013 / 615745(2013).

[0428] Hammond,T.R.et al.Single-Cell RNA Sequencing of Microglia throughoutthe Mouse Lifespan and in the Injured Brain Reveals Complex Cell-StateChanges.Immunity50,253-271 e256,doi:10.1016 / j.immuni.2018.11.004(2019).

[0429] Bennett,M.L.et al.New tools for studying microglia in the mouse andhuman CNS.Proc NatlAcad Sci U S A113,E1738-1746,doi:10.1073 / pnas.1525528113(2016).

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[0433] Robinson,M.D.,McCarthy,D.J.&Smyth,G.K.edgeR:a Bioconductor packagefor differential expression analysis of digital gene expressiondata.Bioinformatics 26,139-140,doi:10.1093 / bioinformatics / btp616(2010).

[0434] McCarthy,D.J.,Chen,Y.&Smyth,G.K.Differential expression analysis ofmultifactor RNA-Seq experiments with respect to biological variation.NucleicAcids Res40,4288-4297,doi:10.1093 / nar / gks042(2012).

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[0437] Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82, 239-259, doi: 10.1007 / BF00308809 (1991).

[0438] Braak, H., Alafuzoff, I., Arzberger, T., Kretzschmar, H. & Del Tredici, K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol 112, 389-404, doi: 10.1007 / s00401-006-0127-z (2006).

[0439] Example 3

[0440] Example 3 - Characteristics of Angiopep-2Ab

[0441] Although antibodies have been modified by many approaches to address the bottleneck problem of delivery into various tissues, approaches to penetrate the blood-brain barrier (BBB) ​​currently represent the most difficult hurdle. One approach is based on the conjugation of Angiopep-2 peptides to host proteins, including immunoglobulins, to increase penetration across brain endothelial cells. In principle, receptor-dependent transcytosis of Angiopep-2 might facilitate the passage of its cargo across the BBB (via interaction with low-density lipoprotein receptor-related protein 1 [LRP-1])1. However, in our hands, this approach has been unsatisfactory in terms of its effects on brain penetration and on the ability of transcytotic antibodies to bind antigens in the brain.

[0442] Although several patents use Angiopep-2 to accelerate the penetration of small molecules or multiple peptides into the brain, we were able to identify only one patent (U.S. Pat. No. 9161988) that used angiopep-2Ab conjugates to enhance BBB penetration. We tested this approach based on brain penetration of labeled Angiopep-conjugated Abs and the antigen binding capacity of the penetrating Abs (these issues were not addressed in the aforementioned U.S. Pat. No. 9161988). We noted relatively weak activity ( Fig.21). We reasoned that modification of the positive charge of Angiopep2-Ab might enhance interaction with negatively charged endothelial cells in the brain. Without being bound by theory, increasing the flexibility of the Angiopep-2-Ab conjugation bridge could reduce its aggregation potential and make the conjugated Ab more functional. These considerations led to a solution to this problem that achieves the features discussed below. In brief, we designed a KK modification and Ahx linker to enhance brain penetration of Angiopep2-conjugated mAbs.

[0443] First, we added 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 being bound by theory, this enhances penetration into the BBB via adsorption-mediated transport (AMT), which depends on the interaction between the negatively charged surface of endothelial cells and the positively charged substituted ligand.

[0444] Second, we added the Ahx linker to the Angiopep2 sequence. Ahx can increase the flexibility of the peptide chains and keep them properly solvated to prevent aggregation and the associated receptor-mediated transcytosis (RMT) and reduction in the functional penetration of Angiopep-2Ab ( Fig. 22 ).

[0445] References cited in this example

[0446] Demeule.M.et al.Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2.JNeurochem 106, 1534-1544, doi: 10.11 11 / j.1471-4159.2008.05492.x (2008).

[0447] Example 4

[0448] Example 4 - Development of engineered brain-penetrating monoclonal antibodies targeting osteopontin (OPN) for Alzheimer's disease therapy Cloned Antibodies (mAbs)

[0449] Recent clinical trials of anti-amyloid-β (Aβ) antibodies have reported significant reductions in plaque burden, but no clear improvements in cognitive performance. These disappointing results have accelerated efforts to identify and validate novel drug targets that can halt or reverse disease progression and cognitive decline. Recently, Aβ plaques have been divided into two subpopulations: a diffuse, aggressive neurotoxic plaque subpopulation that impairs cognition, and a dense subpopulation that likely represents a nontoxic product of microglial processing and compaction of Aβ fibrils (Huang et al., 2021; Parhizkar et al., 2019; Yuan et al., 2016). If microglial packaging of Aβ fibrils into dense plaques is a neuroprotective mechanism, general disaggregation of dense plaques by antibodies may be counterproductive. These considerations also suggest that therapeutic enhancement of microglial plaque compaction may represent a more effective therapeutic strategy compared to current approaches that indiscriminately target plaques in mAb trials.

[0450] Based on preclinical studies using the 5XFAD model of Alzheimer's disease (AD) and analysis of a well-characterized set of human brain tissue, we have defined the Spp1 gene (encoding osteopontin, OPN) as a novel and tractable target. We have also developed strategies to test the therapeutic potential of blood-brain barrier (BBB)-penetrating antibodies specific for OPN. This anti-OPN mAb can improve cognitive function in 5XFAD mice by reducing neurotoxic diffuse Aβ plaques and microglial inflammatory responses.

[0451] Defining OPN as a therapeutic target for AD :

[0452] Although many disease-associated genes have been identified in murine models of AD, most have not been confirmed from studies of human AD. 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 have shown that genetic deletion of OPN in 5XFAD mice substantially reduces inflammatory microglia, amyloid-β (Aβ) plaque area, and significantly improves cognitive function. Our mechanistic studies suggest that targeting OPN inhibits the TREM2-dependent pathway responsible for microglial uptake of Aβ fibrils, lysosomal digestion, and extrusion into the brain parenchyma (Huang et al., 2021; Keren-Shaul et al., 2017; Parhizkar et al., 2019; Yuan et al., 2016), to reduce the spread of toxic Aβ oligomers throughout the brain.

[0453] We have used brain tissues (Sinai Mountain Brain Bank) from clinical and neuropathological characterizations of AD patients and controls to test the validity of these rodent findings in human disease. The production of OPN by human microglia is closely related to the severity of AD and the pre-mortem assessment of cognitive impairment, as judged by the comparison of brain materials from AD patients and healthy controls (in collaboration with Dr. Michal Beeri of Mount Sinai). The increase in the number of microglia producing OPN from AD brain slices is closely related to progressive dementia, as measured by the Clinical Dementia Rating (CDR) scale. Based on these studies, the blockade of OPN can reduce proinflammatory microglia, promote lysosomal degradation and the detoxification of related Aβ plaques, thereby improving cognitive function.

[0454] Generation and testing of monoclonal anti-OPN Abs engineered for enhanced brain penetration :

[0455] We have generated a fluorescently labeled (AF488) mAb after conjugating a monoclonal anti-OPN Ab (MPIIIB10) to a chemically modified Angiopep-2 peptide (Ang2pep). Ang2pep mediates receptor-dependent transcytosis and crosses the blood-brain barrier (BBB) ​​via interaction with low-density lipoprotein receptor-related protein 1 (LRP-1) expressed by brain endothelial cells. This strategy will allow OPN blockade for in vivo therapeutic purposes.

[0456] What are the characteristics of the disclosed method? Compared with existing methods, what improvements or advantages does the disclosed method have?

[0457] Our approach is based on a more precise definition of the contribution of OPN, which has been identified as part of a gene signature in animal models of AD and in human disease. Targeting OPN with a monoclonal Ab can reduce Aβ pathology and improve cognitive function, in stark contrast to current Aβ-based approaches that indiscriminately target all Aβ plaques. We have engineered a brain-penetrating anti-OPN mAb to selectively target neurotoxic Aβ plaques for proof-of-principle studies and as a basis for the development of new and effective antibody-based AD therapeutics.

[0458] Several features of OPN enhance the clinical feasibility of targeting this protein by mAb. OPN is an extracellular protein secreted by microglia that is easily accessible to mAb. OPN production is generally associated with pathogenic responses rather than protective responses. OPN expression in peripheral tissues contributes to several chronic disorders, including atherosclerosis and cardiovascular disease, autoimmune diseases, and cancer growth and metastasis, and genetic or antibody-based reduction of OPN can improve pathology in these clinical settings. In contrast, genetic deletion or reduction of OPN does not suppress general immune responses.

[0459] What are the potential applications of the disclosed method? How will these applications be carried out?

[0460] OPN expression significantly increases pro-inflammatory microglia and inhibits lysosomal degradation and associated detoxification of Aβ plaques. Effective targeting of OPN by anti-OPN mAbs is applicable to AD and other OPN-mediated neuroinflammatory diseases, such as multiple sclerosis (MS).

[0461] Since OPN expression is upregulated in microglia during disease progression and persistent AD, antibody-based neutralization of OPN is during a time window when OPN-mediated effects are most damaging and pathogenic.

[0462] What other methods can be used in combination with the disclosed methods?

[0463] The following approaches can be used to additionally enhance brain penetration of anti-OPN mAbs:

[0464] 1. Cell penetrating peptides (CPP) are a group of short peptides, consisting of amphipathic and / or cationic sequences that allow passage through the cell membrane. For example, the transactivator protein (TAT) of transcription peptides is an effective CPP that has been shown to effectively transport various therapeutic agents to the brain of a mouse model with different CNS disorders. Positively charged TAT can promote interaction with negatively charged cell membranes at the BBB, and trigger internalization by adsorption-mediated transcytosis (AMT). This represents a receptor / transporter-independent pathway different from the Ang2pep pathway. If necessary, we will construct the mAb of the dual conjugation of Ang2pep-TAT to determine whether the Ab conjugates utilizing both receptor-mediated transcytosis (RMT) and adsorption-mediated transcytosis (AMT) pathways will allow increased penetration and higher Ab concentrations in brain tissue.

[0465] 2. A two-stage injection regimen can be used to maximize brain penetration of anti-OPN mAbs. Unconjugated ("cold") anti-OPN mAb can be injected at time 0, which will allow occupation of available OPN and Fc receptors expressed by peripheral tissues prior to injection of Angiopep2-conjugated ("hot") anti-OPN mAb, thereby enhancing brain penetration of the conjugated "hot" Ab.

[0466] Describe additional research plans .

[0467] We have 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, leading to increased aggregation and extrusion of non-toxic Aβ plaques. These changes were accompanied by significant improvements in cognitive function. We have engineered brain-penetrating peptide-conjugated anti-OPN mAbs to test their therapeutic effects in vivo using an AD mouse model. Analysis of brain penetration and therapeutic activity of the conjugated anti-OPN mAb will depend on comparisons between OPNWT.5XFAD mice and OPN- / -.5XFAD mice to distinguish specific anti-OPN binding to brain OPN and nonspecific uptake of anti-OPN mAb (MPIIIB1 0).

[0468] 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 will be harvested, and the intensity of the AF488 fluorophore will be measured using a fluorescent microplate reader to obtain raw fluorescence units (RFUs). Antibody concentrations will be quantified as a percentage of injected dose per gram of tissue (%ID / g): %ID / g = RFU measured per gram of brain (or tissue) / total RFU injected. In addition, brain capillary depletion assays will be performed to distinguish Ang2pep-mediated transcytosis of Abs to the brain interstitium and binding to the brain microvasculature based on the intensity of the fluorophore (AF488) in the capillary-enriched fraction and the parenchyma fraction of brain homogenates at different time points after Ab injection. The binding specificity of the conjugated Ab at the optimized dose was also determined by counter staining of OPN brain sections from 5XFAD mice and OPN- / -.5XFAD mice with a secondary fluorophore following injection of the conjugated Ab.

[0469] The therapeutic activity of the anti-OPN mAb conjugated using the optimized dosage regimen will be evaluated for the impact on Aβ plaque pathology and neuroinflammation according to the number of Aβ plaques / area, plaque compaction and TNF-α+ microglia. The Aβ plaque load will be checked by the immunofluorescence of hippocampus and cortical slices with anti-Aβ Ab6E 1 0, and confirmed by Aβ positron emission tomography (PET) imaging according to the average PET standard uptake value ratio (SUVR) comprehensive score of the 18F-Florbetapir tracer combined with Aβ plaques. Microglial lysosomal activation (CD68 lysosomal activation protein and cathepsin B enzyme) will be assessed by the immunofluorescence of mouse brain cryosections. The impact of anti-OPN mAb on neuropathology will be determined according to the number of dystrophic neurites with labeled anti-APP Ab, and if the above analysis is required, it will be further confirmed by using T-type water maze and Y-type Novel maze to check cognitive function.

[0470] References cited in this example :

[0471] 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 andengulf amyloid beta plaques. Nat Immunol.

[0472] 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 169,1276-1290e1217.

[0473] 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.

[0474] Yuan, P., Condello, C., Keene, CD, Wang, Y., Bird, TD, Paul, SM, Luo, W., Colonna, M., Baddeley, D., and Grutzendler, J. (2016). TREM2Haplodeficiency in Miceand Humans Impairs the Microglia Barrier Function Leading to DecreasedAmyloid Compaction and Severe Axonal Dystrophy.Neuron 92, 252-264.

[0475] Example 5

[0476] Example 5 - Development of engineered brain-penetrating monoclonal antibodies targeting osteopontin (OPN) for Alzheimer's disease therapy Cloned Antibodies (mAbs)

[0477] An exemplary strategy for developing engineered brain-penetrating monoclonal antibodies targeting OPN for Alzheimer's disease therapy is shown below.

[0478] In a working model, OPN's inhibition of microglial Aβ plaque compaction and promotion of inflammatory responses led to cognitive impairment. OPN was defined as a therapeutic target for Alzheimer's disease (AD) from: i) data from the 5XFAD mouse model, and ii) data from AD patients and controls (Mount Sinai Brain Bank). In addition, monoclonal anti-OPN Abs were engineered to enhance brain penetration.

[0479] An exemplary working model of microglial OPN expression is shown in Fig.23In this model, OPN expression promotes microglial proinflammatory responses (TNF-α) and inhibits the TREM2 / Axl / lysosomal engulfment pathway, thereby inhibiting lysosomal degradation and Aβ plaque compaction, which ultimately leads to cognitive impairment. The OPN-based platform described herein targets diffuse Aβ plaques and microglial proinflammatory responses to simultaneously inhibit two disease-exacerbating factors.

[0480] Microglia are the major cellular source of OPN production in the brain of 5XFAD mice. As shown in Figure 24, microglial expression of OPN was increased at both mRNA and protein levels in 5XFAD mice compared to age-matched WT mice with disease progression.

[0481] In the brain of 5XFAD mice, OPN expression is restricted to CD11c + During disease progression, CD11c in 5XFAD mice was upregulated compared with age-matched WT mice. + OPN + The percentage of microglia increased significantly (Figure 25).

[0482] Genetic deletion of OPN reduced microglial TNF-α production, total Aβ plaque area, the ratio of diffuse plaques, and the number of dystrophic neurites, leading to the rescue of cognitive impairment ( FIG. 26 ).

[0483] CD11c + Transcriptomic analysis of microglia showed that OPN deficiency led to downregulation of genes involved in proinflammatory responses and upregulation of genes involved in phagocytosis. Genetic deficiency of OPN reduced αVβ3 + CD11c + TNF-α production by microglia without reduction of αVβ3-CD11c + TNF-α production by microglia, indicating that OPN promotes microglial proinflammatory responses via its interaction with αVβ3 integrin receptors. In addition, in situ analysis of 5XFAD mouse brain cryosections showed that OPN deficiency increased microglial phagocytosis of Aβ (Figure 27).

[0484] TREM2 is exclusively expressed by CD11c in the brain of 5XFAD mice + Microglia express. Genetic deletion of OPN results in CD11c + Increased TREM2 expression by microglia and activation of lysosomes, as indicated by CD11c, CD68, and cathepsin B in OPN-KO.5XFAD mice compared to 5XFAD mice + This suggests that OPN inhibits CD11c expression in 5XFAD mice, as judged by increased microglial expression. +TREM2 lysosomal engulfment pathway in microglia ( FIG. 28 ).

[0485] OPN deficiency resulted in a significant decrease in the total area of ​​Aβ plaques (6E 1 0 + ) and the increase in compacted plaque area (6E10 + Thio-S + ), such as by the patch compactness index (6E10 + Thio-S + Area / 6E10 + This indicates that OPN inhibits the expression of CD11c in Aβ plaques. + Microglia were compacted (Figure 29).

[0486] Measurement of OPN in the homogenate of the middle frontal gyrus showed that OPN expression in the brains of AD patients increased 3-fold compared to cognitively normal control subjects. Increased OPN expression in the brains of AD patients was also noted compared to the brains of patients with MCI, 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).

[0487] We determined the percentage of CD11c+OPN+ (double positive, DP) microglia by immunofluorescence analysis of human brain sections from the middle frontal gyrus, an area affected by AD early on. Normal controls and MCI subjects showed similar proportions of CD11c+OPN+ microglia (CD11c+OPN+Iba-1+). We observed a 3-fold increase in CD11c+OPN+ microglia in brain sections from AD patients compared to sections from normal controls. In addition, 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 represents a sensitive parameter for distinguishing AD patients from patients with MCI. This conclusion is further supported by the very strong correlation between the percentage of CD11c+OPN+ microglia and the CDR score (Figure 31).

[0488] Higher brain OPN levels were associated with higher levels of neuritic plaques (r=0.4919, p=0.0043). Similar to the association with dementia severity, there was a stronger 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 can be a sensitive marker for both clinical severity and neuropathology of AD (Figure 32).

[0489] We conjugated Angiopep2 to anti-CD11b mAb and tested the binding activity of 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 did not change the binding activity of mAb (Figure 33).

[0490] We conjugated a modified Angiopep2 peptide (containing a -KK- amino acid bridge and an AhX linker) to an anti-CD11b mAb. In vivo testing showed effective brain penetration of the conjugated anti-CD11b mAb. Angiopep-2 conjugated Ab showed a 10-fold increase ( Fig.34 ).

[0491] Microglia isolated from 9-month-old 5XFAD mice were incubated with increasing concentrations (5 μg / ml, 10 μg / ml, 20 μg / ml) of anti-OPN mAb (MPIIIB10) for 24 h, followed by CD11c + Microglial TNF- α Flow cytometry analysis was performed. Microglia incubated with isotype control (mouse IgG 1) served as a negative control. Anti-OPN mAb blockade resulted in the suppression of CD11c + Microglial TNF- α The dose-dependent inhibition ( Fig.35 ).

[0492] Example 6

[0493] Example 6 - Exemplary Methods of Treating Alzheimer's Disease

[0494] Our approach to Alzheimer's disease is based in part on the following:

[0495] Unmet needs: i) Dementia / Alzheimer's disease affects 55 million people (2020) and is expected to affect 130 million people by 2050; ii) There are no effective treatments. Anti-Aβ antibodies (e.g., aducanumab) reduce plaques but do not improve cognition ($55,000 / year); and iii) The impact of long-term care on families, healthcare systems, and society.

[0496] OPN-based therapeutics: Engineered brain-penetrating anti-OPN mAbs selectively remove toxic plaques and improve cognitive function.

[0497] Schematic diagram ( Fig.36) shows OPN-dependent regulation of protective Aβ degradation and compaction by microglia. In the OPN-low brain, microglia take up and degrade Aβ plaques in activated lysosomes, subsequently extruding non-toxic compacted plaques into the microenvironment. In the OPN-high AD brain, OPN inhibits microglial uptake and compaction of toxic diffuse plaques while promoting proinflammatory responses (TNF-α production). Blocking OPN with anti-OPN mAb or integrin inhibitors inhibits the proinflammatory response of microglia and enhances microglial uptake and compaction of diffuse plaques ( Fig.36 ).

[0498] Fig.37 The schematic diagram in Figure 2 shows an exemplary OPN mechanism of action: We identified a pathogenic CD11c + Microglial subpopulations that promote proinflammatory responses and inhibit microglial uptake / compaction of Aβ plaques, thereby contributing to AD pathology and cognitive impairment ( Fig.37 ).

[0499] Genetic deletion of OPN results in neurotoxic plaques (6E10 + Thio-S - ) and the number of dystrophic neurites, which ultimately rescued cognitive impairment in 5XFAD mice ( Fig.38 ).

[0500] In an AD mouse model (5XFAD mice), OPN deficiency leads to a significant reduction in toxic diffuse plaques and 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 plaques, and tau tangles ( Fig.39 ).

[0501] We conjugated brain-delivered Abs containing the Angiopep2 peptide, a -KK-amino acid bridge, and an AhX linker to anti-CD11b mAb. In vivo testing showed effective brain penetration of the conjugated brain-delivered anti-CD11b mAb. Angiopep2-conjugated Abs showed approximately a 2-fold increase in fluorescent microglia compared to unconjugated anti-CD11b Abs. KK-Ahx-modified Angiopep-2 Abs showed a 2- to 3-fold increase compared to Angiopep-2-conjugated Abs, and a 10-fold increase compared to unconjugated anti-CD11b Abs ( Fig.40 ).

[0502] The first generation of conjugated anti-OPN mAb for brain delivery required the conjugation of modified Angiopep2 (containing a -KK- bridge and an AhX linker) to the anti-OPN mAb. Another conjugation strategy could generate an Angiopep2-TAT dual conjugate, which could further increase the brain penetration of the anti-OPN mAb ( Fig.41 ).

[0503] Example 7

[0504] Example 7 - Administration of anti-OPN mAb inhibits microglial proinflammatory responses and improves Aβ plaque pathology

[0505] Anti-OPN was administered weekly to 6-month-old 5XFAD mice for 1 or 2 months. Microglial proinflammatory responses and Aβ plaque pathology were analyzed after 1 or 2 months of treatment ( FIG. 42A ).

[0506] Compared to control mice, one month of treatment resulted in a modest reduction in CD11c+ microglia, and two months of treatment resulted in a 45% reduction in CD11c+ microglia. Compared to isotype-matched control mice, one month of treatment resulted in a 35% reduction in TNF-α expression by CD11c+ microglia, and two months of treatment resulted in a 55% reduction in TNF-α expression by CD11c+ microglia (Figure 42B).

[0507] Representative immunofluorescence images of brains from 5XFAD mice after 1 or 2 months (FIG. 42C and FIG. 42D). The first column of images in (C) and (D) was stained with 6E10 to identify diffuse and aggregated forms of Aβ plaques. The second column of images was stained with Thioflavin-S to identify only β-sheets + Aβ aggregated plaques. The third column shows the merged image of the first and second columns. Quantification of total plaque area, diffuse plaque area, and plaque compactness index after 1 and 2 months of treatment is shown in (EG).

[0508] Example 8

[0509] Example 8 - Administration of cyclic RGD (cilengitide) inhibits microglial proinflammatory responses

[0510] Cyclic RGD (cilengitide) was administered intravenously or intranasally weekly to 6-month-old mice for 1 or 2 months. Microglial proinflammatory responses and Aβ plaque pathology were analyzed after 1 or 2 months of treatment ( FIG. 43A ).

[0511] Compared with control mice, intranasal administration of Cilengitide for one month resulted in a decrease in CD11c+ microglia, and two months resulted in a 35% decrease in CD11c+ microglia. Compared with control mice, intranasal administration of Cilengitide for one month resulted in a decrease in TNF-α expression of CD11c+ microglia by about 35%, and two months resulted in a decrease in TNF-α expression of CD11c+ microglia by about 45% (Figure 43B and Figure C).

[0512] Compared with control mice, intravenous administration of cilengitide for one month resulted in a 50% decrease in CD11c+ microglia, and two months resulted in a 60% decrease in CD11c+ microglia. Compared with control mice, intravenous administration of cilengitide for one month resulted in a decrease in TNF-α expression by CD11c+ microglia by about 50%, and two months resulted in a 55% decrease in TNF-α expression by CD11c+ microglia (Figure 43B and Figure C).

[0513] Example 9

[0514] Example 9 - Intranasal Administration of Antibodies

[0515] Typically, in many of the previously described studies, administration of Angiopep-2-conjugated antibodies was performed via the intravenous route. Without wishing to be bound by theory, since osteopontin is present in a variety of tissues, these tissues may serve as a "sink" for anti-OPN antibodies when the antibodies are administered systemically (e.g., intravenously).

[0516] In the studies described in this example, intranasal administration of conventional antibodies (i.e., antibodies that are not conjugated to a moiety for delivering an active / therapeutic agent such as Angiopep-2) was tested as a way to increase delivery of the antibody to the brain, potentially avoiding the "sink" effect seen when Angiopep-2-conjugated antibodies are administered intravenously. Without being bound by theory, intranasal delivery was tested as a way to circumvent the blood-brain barrier by using olfactory transfer to deliver agents to the brain.

[0517] Figure 44A to Figure 44C show the intranasal delivery of conventional unconjugated anti-CD11b monoclonal antibodies. Antibodies (10 mg / kg body weight) were delivered intranasally to 6-month-old 5XFAD mice (n=4) at time 0. The antibodies were labeled with the fluorophore AF488. Observations were obtained 3 hours after administration. The control was an intravenous injection of anti-CD11b monoclonal antibodies (also unconjugated). (A) shows the anti-CD11b monoclonal antibody fluorescence (green) in the brain administered intranasally compared to the control intravenous administration. (B) shows the anti-CD11b monoclonal antibody fluorescence (green) in the brain administered intranasally, and also shows the immunofluorescence (red) of anti-Iba-1 antibodies. Iba-1 is a microglia marker. The combined immunofluorescence (yellow) confirms the binding specificity of anti-CD11b monoclonal antibodies to microglia. (C) shows data from these studies calculating the penetration of anti-CD11b mAbs into the brain, which was calculated as the number of fluorescent microglia per microscopic field (μm) for anti-CD11b mAbs administered via the intranasal route compared to intravenous administration. 2 ×10 6 ). The data indicated that intranasal administration of the antibody resulted in approximately 10-fold increase in brain penetration compared to intravenous injection.

[0518] Example 10

[0519] Example 10- Effect of OPN on the activation of microglial inflammasomes

[0520] Activation of the NLRP3 (pyrin domain-containing NLR family 3) inflammasome represents the main neuroinflammatory component of AD pathology in human and mouse Alzheimer's disease (AD) models. We analyzed the effect of OPN on microglial inflammasome activation in vitro based on caspase-1 activity and IL-1β production. Microglial intracellular caspase-1 activity was determined using a bioluminescent assay that specifically detects caspase-1 activation, which was confirmed by a selective caspase-1 inhibitor (Figure 45A). We observed that recombinant mouse OPN (rmOPN) significantly increased caspase-1 activity and IL-1β production in LPS+Aβ-stimulated microglia from both 9-month-old 5XFAD and OPN-KO.5XFAD mice. This OPN-dependent promotion of inflammasome activation was blocked by an αVβ3 inhibitor ( FIGS. 45B-45C ), suggesting that OPN-dependent promotion of microglial inflammasome activation may reflect its interaction with the αVβ3 integrin.

[0521] In these studies, microglia isolated from 9-month-old 5XFAD and OPN-KO.5XFAD mice were cultured at 6 × 10 cells / mL in conventional microglial culture medium (DMEM-F12 with 10% fetal bovine serum + 1% penicillin / streptomycin + 10 ng / ml recombinant mouse M-CSF). 4 Cells / well were seeded into 96-well plates. To induce inflammasome activation, microglia were primed with 100 ng / ml LPS for 3 h and then stimulated with 10 μM Aβ1-42 fibrils overnight in the presence or absence of 12.5 μg / ml rmOPN. αVβ3 inhibitor (cilengitide, Selleck, 10 μM) was added to the culture medium 1 h before the addition of rmOPN. Caspase- 1 Inflammasome Assay Kit (Promega) was used to analyze the intracellular caspase-1 activity of microglia. The selective caspase-1 inhibitor (Ac-YVAD-CHO, 1 μM) included in the kit was used to verify the detection specificity of caspase-1 activity. The culture medium was collected for quantitative determination of microglial IL-1 β production by mouse IL-1 β DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0522] *****

[0523] Equivalent

[0524] 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 described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the appended claims.

[0525] Table 1: DEGs of CD11c+ microglia in 9-month-old OPN-KO.5XFAD and 5XFAD

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[0591] Table 2: Overview of human samples included in this study.

[0592]

[0593] PMI, postmortem interval (minutes); CDR, clinical dementia rating; IF, immunofluorescence staining

[0594] Age- and sex-matched samples were analyzed for OPN levels as measured by ELISA on frozen samples followed by immunofluorescence staining for 1ba-1 / CD11c / OPN (fixed, paraffin-embedded sections).

[0595] Table 3: Characterization of frozen and fixed human samples.

[0596]

[0597]

[0598] 1 Determination was performed post-mortem by a neuropathologist before storage of samples in the Mount Sinai Brain Bank. PMI: post-mortem interval (minutes); CDR: clinical dementia rating; IF: immunofluorescence staining.

Claims

1. A compound capable of crossing the blood-brain barrier of a patient, the compound comprising an active agent conjugated to a bridge, linker, carrier agent, or a combination thereof; The carrier agent is capable of crossing the blood-brain barrier.

2. A moiety for delivering an active agent across the blood-brain barrier of a patient, the moiety comprising: bridge, linker and carrier agents; wherein the carrier agent is capable of crossing the blood-brain barrier and the bridge is capable of crossing the blood-brain barrier.

3. The compound of claim 1 or the moiety of claim 2, wherein the active agent comprises a therapeutic agent, a cytotoxic agent, or an imaging agent.

4. The compound of claim 1 or the moiety of claim 2, wherein the active agent comprises an αVβ3 inhibitor.

5. The compound according to claim 4, wherein the αVβ3 inhibitor comprises a small molecule containing cyclic RGD or a derivative thereof, including cilengitide or a derivative thereof.

6. The compound of claim 3, wherein the active agent comprises a protein or a polypeptide.

7. The compound of claim 4, wherein the active agent comprises a monospecific, bispecific or multispecific antibody.

8. The compound of claim 6, wherein the active agent 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.

9. The compound of claim 6, wherein the active agent comprises a single-chain antibody.

10. The compound of claim 9, wherein the antibody comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

11. The compound of claim 1 or the moiety of claim 2, wherein the active agent comprises an anti-osteopontin (OPN) antibody.

12. The compound of claim 1 or the moiety of claim 2, wherein the active agent comprises an anti-CD11b antibody.

13. The compound of claim 1 or the moiety of claim 2, wherein the carrier agent comprises a peptide.

14. The compound of claim 13, wherein the peptide comprises a cell penetrating peptide or a cell targeting peptide.

15. The compound of claim 13, wherein the peptide is about 30 amino acids in length.

16. The compound of claim 13, 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-EE (SEQ ID NO: 7), GSH, HSTPSSP (SEQ ID NO: 8), DSSLFAL (SEQ ID NO: 9), YGRKKRRQRRRPPQQ (SEQ ID NO: 10), LLIILRRRIRKQAHAHSK (SEQ ID NO: 11), RRLSYSRRRF (SEQ ID NO: 12), or any combination thereof.

17. The compound of claim 13, wherein the carrier agent comprises Angiopep-2.

18. The compound of claim 13, wherein the carrier agent is capable of crossing the blood-brain barrier.

19. The compound of claim 13, wherein the carrier agent is capable of crossing the blood-brain barrier via receptor-dependent transcytosis.

20. The compound of claim 1 or the moiety of claim 2, wherein the bridge comprises an amino acid.

21. The compound of claim 20, wherein the bridge is positively charged.

22. The compound of claim 20, wherein the bridge comprises arginine, lysine, histidine, or a combination thereof.

23. The compound of claim 20, wherein the bridge comprises at least two consecutive lysines.

24. The compound of claim 20, wherein the bridge is capable of crossing the blood-brain barrier.

25. The compound of claim 20, wherein the bridge is capable of crossing the blood-brain barrier via adsorption-mediated transcytosis.

26. The compound of claim 20, wherein the bridge is capable of increasing the isoelectric point of the compound or moiety.

27. The compound of claim 1 or the moiety of claim 2, wherein the linker comprises an amino acid.

28. The compound of claim 27, wherein the linker comprises a flexible linker.

29. The compound of claim 28, wherein the flexible linker is capable of increasing the flexibility of the moiety or compound.

30. The compound of claim 28, wherein the flexible linker is capable of facilitating solvation of the compound or moiety.

31. The compound of claim 28, wherein the flexible linker is capable of reducing aggregation of the compound or moiety.

32. The compound of claim 28, wherein the flexible linker comprises a small polar amino acid or a non-polar amino acid.

33. The compound of claim 32, wherein the small polar amino acid comprises Ser or Thr.

34. The compound of claim 32, wherein the non-polar amino acid comprises Gly.

35. The compound of claim 32, wherein the flexible linker comprises Ser, Thr, Gly, or a combination thereof.

36. The compound of claim 28, 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).

37. The compound of claim 36, wherein the flexible linker comprises 6-aminohexanoic acid (Ahx).

38. The compound of claim 26, wherein the linker is capable of providing a spacer between the active agent, carrier agent, or bridge.

39. The compound of claim 1 or the moiety of claim 2, further comprising a conjugate linking the active agent to the bridge, linker or carrier agent.

40. The compound of claim 39, wherein the conjugate comprises -N-hydroxysuccinimide-ester (-NHS ester).

41. The compound of claim 40, wherein the -NHS-ester comprises a monofluoro-substituted cyclooctyne (MFCO)-NHS-ester, a sulfo-NHS-ester, or a biotin-NHS-ester.

42. A pharmaceutical composition for treating a neurodegenerative disease in a patient, the pharmaceutical composition comprising an anti-osteopontin (OPN) antibody, wherein the pharmaceutical composition comprises means for crossing the blood-brain barrier.

43. The pharmaceutical composition according to claim 42, further comprising an αVβ3 inhibitor.

44. The pharmaceutical composition of claim 43, wherein the αVβ3 inhibitor comprises cilengitide.

45. The pharmaceutical composition of claim 42, wherein the neurodegenerative disease comprises Alzheimer's disease, multiple sclerosis, Parkinson's disease, or amyotrophic lateral sclerosis.

46. ​​A pharmaceutical composition comprising the compound according to claim 1.

47. The pharmaceutical composition of claim 42, wherein the means for crossing the blood-brain barrier comprises a portion for delivering an active agent, the portion comprising: bridge, linker and carrier agents; wherein the carrier agent is capable of crossing the blood-brain barrier and the bridge is capable of crossing the blood-brain barrier.

48. A method of delivering an active agent to the central nervous system of a patient in need thereof, the method comprising: administering peripherally the compound of claim 1; as well as The active agent is allowed to cross the blood-brain barrier.

49. A method of delivering an active agent to the central nervous system of a patient in need thereof, the method comprising: Intranasal administration of the compound according to claim 1; as well as The active agent is allowed to enter the central nervous system of the patient.

50. The method according to one of claims 48 or 49, for treating a neurodegenerative disease in a patient.

51. The method of claim 50, wherein the neurodegenerative disease comprises Alzheimer's disease or multiple sclerosis.

52. A method for treating Alzheimer's disease in a patient, the method comprising administering a therapeutic agent capable of targeting CD11c+OPN+ microglia.

53. The method of claim 52, wherein the therapeutic agent is capable of depleting the CD11c+OPN+ microglia.

54. The method of claim 52, wherein the therapeutic agent comprises an anti-osteopontin antibody.

55. The method of claim 52, wherein the therapeutic agent comprises an αVβ3 inhibitor.

56. The method of claim 52, wherein the therapeutic agent comprises an anti-osteopontin antibody in combination with an αVβ3 inhibitor.

57. The method of claim 52, wherein the therapeutic agent comprises an active agent conjugated to a bridge, a linker, a carrier agent, or a combination thereof.

58. The method of claim 52, wherein the therapeutic agent is administered intranasally.

59. The compound of claim 1 comprising the structure of Figure 22.

60. A therapeutic compound for use in treating Alzheimer's disease, the therapeutic compound comprising: Anti-osteopontin (OPN) antibodies; and The moiety conjugated to the antibody comprises an amino acid bridge, a flexible amino acid linker and a peptide carrier agent.

61. The therapeutic compound of claim 60, wherein the amino acid bridge comprises Lys-Lys.

62. The therapeutic compound of claim 60, wherein the flexible amino acid linker comprises 6-aminohexanoic acid.

63. The therapeutic compound of claim 60, wherein the peptide carrier agent comprises Angiopep-2.

64. The therapeutic compound of claim 63, wherein the Angiopep-2 has the amino acid sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1) or an amino acid sequence that is 90% identical to SEQ ID NO:

1.

65. The therapeutic compound of claim 60, wherein the moiety is conjugated to the antibody via MFCO-N-hydroxysuccinimide ester.

66. The therapeutic compound according to any one of claims 60 to 65, which has a linear arrangement, in the order: anti-osteopontin antibody-conjugate-amino acid bridge-flexible amino acid linker-peptide carrier agent.

67. The therapeutic agent according to claim 66, which has a linear arrangement, in order: anti-osteopontin antibody-monofluoro-substituted cyclooctyne-N-hydroxysuccinimide ester conjugate-Lys-Lys amino acid bridge-6-aminohexanoic acid flexible linker-Angiopep-2 peptide carrier agent.

68. A method for designing a therapeutic compound for treating a neurodegenerative disease in a patient, the method comprising: selecting monoclonal antibodies capable of depleting cells contributing to said neurodegenerative disease; The components of the moiety are selected to be conjugated to the monoclonal antibody, the components of the moiety comprising: a peptide carrier agent capable of crossing the blood-brain barrier via receptor-dependent transcytosis; a positively charged amino acid bridge selected to raise the isoelectric point (pi) of the therapeutic compound to basic levels; a flexible amino acid linker selected to increase flexibility, promote solvation, or reduce aggregation of the therapeutic compound; as well as The conjugate is selected to attach the moiety to the monoclonal antibody.

69. A method for delivering an active agent to the brain of a subject, the method comprising: The active agent is administered via the intranasal route.

70. The method of claim 69, wherein the active agent is administered in the form of a nasal spray.

71. The method of claim 69, wherein the active agent comprises, consists essentially of, or consists of a small molecule.

72. The method of claim 69, wherein the active agent comprises, consists essentially of, or consists of a peptide.

73. The method of claim 72, wherein the peptide comprises, consists essentially of, or consists of a cyclic peptide.

74. The method of claim 69, wherein the active agent comprises, consists essentially of, or consists of a protein.

75. The method of claim 74, wherein the active agent comprises, consists essentially of, or consists of an antibody.

76. The method of claim 75, wherein the antibody comprises, consists essentially of, or consists of: Monoclonal antibody, Fab, F(ab′)2, Fab' single-chain antibody, Fv, single-chain antibody, monospecific antibody, bispecific antibody, trispecific antibody, multivalent antibody, 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.

77. The method of claim 75, wherein the antibody comprises, consists essentially of, or consists of an anti-osteopontin (OPN) antibody.

78. The anti-osteopontin (OPN) antibody of claim 75, wherein the antibody comprises, consists essentially of, or consists of an anti-CD11b antibody.

79. The method of claim 69, wherein the active agent comprises, consists essentially of, or consists of an αVβ3 inhibitor.

80. The method of claim 79, wherein the αVβ3 inhibitor comprises, consists essentially of, or consists of a cyclic RGD-containing small molecule or a derivative thereof, including cilengitide or a derivative thereof.

81. The method of claim 69, wherein the active agent comprises, consists essentially of, or consists of an antibody and an αVβ3 inhibitor.

82. The method of claim 69, wherein the active agent is capable of targeting CD11c+OPN+ microglia.

83. The method of claim 69, wherein the active agent is not conjugated to a moiety for delivery across the blood-brain barrier.

84. A method for treating a neurodegenerative disease in a patient, the method comprising: administering intranasally to the patient a solution comprising an active agent; wherein the active agent is delivered to the brain.

85. The method of claim 84, wherein the active agent is not conjugated to a moiety for delivery across the blood-brain barrier.

86. The method of claim 84, wherein the active agent is not conjugated to a moiety comprising a bridge, a linker, and a carrier agent.

87. The method of claim 84, wherein the active agent comprises, consists essentially of, or consists of an antibody.

88. The method of claim 87, wherein the antibody targets CD11c+OPN+ microglia.

89. The method of claim 84, wherein following intranasal administration, the active agent enters the olfactory epithelial nerve cells in the nasal cavity and moves along the nerve cells into the brain.

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