EP4633661A2 - Compositions and methods of treatment for neuroinflammation-related disorders - Google Patents

Compositions and methods of treatment for neuroinflammation-related disorders

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Publication number
EP4633661A2
EP4633661A2 EP23904757.4A EP23904757A EP4633661A2 EP 4633661 A2 EP4633661 A2 EP 4633661A2 EP 23904757 A EP23904757 A EP 23904757A EP 4633661 A2 EP4633661 A2 EP 4633661A2
Authority
EP
European Patent Office
Prior art keywords
peptides
seq
cns
mhc
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23904757.4A
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German (de)
French (fr)
Inventor
Jonathan Kipnis
Min Woo Kim
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Washington University in St Louis WUSTL
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Washington University in St Louis WUSTL
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Publication of EP4633661A2 publication Critical patent/EP4633661A2/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • the present disclosure generally relates to compositions and methods for the treatment, prevention, or reversal of neuroinflammation-related disorders using fragments of MHC -bound proteins.
  • MHC major histocompatibility complex
  • a pharmaceutical composition which may comprise one or more peptides and a pharmaceutically acceptable excipient.
  • Each of the one or more peptides may bind to a Major Histocompatibility Complex (MHC) molecule and may be a fragment of a protein selected from TABLE Cl, or a modified fragment thereof.
  • Tire protein or peptide may be expressed at elevated levels in the central nervous system (CNS) relative to a control tissue. In one example, an expression level of the protein or peptide is 5 -fold higher than the control tissue. Alternatively, the protein may not be expressed at elevated levels in the CNS or may be expressed at decreased the levels in the CNS relative to a control tissue.
  • the CNS tissue is dural meninges and the control tissue is a lymph node, such as a deep cervical lymph node or a superficial cervical lymph node.
  • Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1- 14619, or a modified sequence thereof. [005]
  • Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294.
  • Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:597. 770, 1231, 1232, 1709, 1748. 2297.
  • Each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, TABLE 8, TABLE 9, or TABLE 10, or a modified sequence thereof.
  • Each of the one or more peptides may be a fragment of a myelin basic protein (MBP). tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM).
  • MBP myelin basic protein
  • TUBB3 tubulin beta 3 class III
  • NEFM neurofilament medium polypeptide
  • Each fragment of MBP may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754. 10755,
  • At least one of the one or more peptides may comprise or consist of the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
  • the pharmaceutical composition may further comprise one or more extracellular vesicles (EV) or liposomes enclosing the one or more peptides.
  • the one or more EV may be an exosome, a microvesicle, or an apoptotic body of a cell, optionally a human cell.
  • the one or more EV may oligodendrocyte- derived. optionally from a dendritic cell, which may be of the cell line DC2.4.
  • the one or more EV may be synthetic.
  • One or more vectors may each comprise at least one of the one or more nucleic acids.
  • the vectors may be viral vectors.
  • Tire viral vectors may be derived from an adenovirus, adeno-associated virus, or retrovirus, optionally selected from a lentivirus.
  • Tire vectors may be non- viral vectors.
  • the one or more nucleic acids may be mRNAs.
  • the mRNAs may be non-replicating mRNAs, self-amplifying mRNAs (saRNAs), or circular RNAs (circRNA).
  • Tire mRNAs may be non-replicating mRNAs; and each rnRNA may comprise a coding region encoding one of the one or more peptides, a first UTR, a second UTR, a 5’ cap. and a poly(A) tail; and each coding region may be flanked by the UTRs.
  • Each 5’ cap may comprise a 7-methylguanosine (m 7 G) cap that is connected to a first nucleotide of each mRNA (N) by triphosphates (ppp) to form m 7 GpppNp.
  • m 7 G cap may comprise a methylated 2 ’-OH on the first nucleotide connecting the 5’ end of each mRNA to the cap (capl or m 7 GpppNimp).
  • Each m 7 G cap may comprise a methylated 2 ’-OH on the first nucleotide and a second nucleotide connecting the 5’ end of each mRNA to the cap (cap2 or m 7 GpppNimpN2mp).
  • Each mRNA may comprise one more modified nucleosides, optionally selected from the group consisting of pseudouridine (t
  • the nucleic acids may be encapsulated, optionally by lipid nanoparticles (LNPs), polyplexes, polymeric nanoparticles, lipopolyplexes (LPPs), or cationic polypeptides.
  • the pharmaceutical composition may comprise the one or more nucleic acids, one or more vectors, or one or more mRNAs.
  • the one or more peptides or the pharmaceutical composition for use in treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury'.
  • a method of treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury in a subject in need thereof which may comprise administering the one or more peptides or the pharmaceutical composition to the subject: and use of the one or more peptides or the pharmaceutical composition in the manufacture of a medicament for ting, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury.
  • Hie peptides or pharmaceutical composition may be or may be intended to be administered to a subject by infusing or injecting into the cerebrospinal fluid (CSF) of the subject.
  • CSF cerebrospinal fluid
  • Hie neuroinflammation-related disorder or an autoimmune neural disorder may be selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN). acute optic neuritis (AON).
  • ADAM acute disseminated encephalomyelitis
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • GBS Guillain-Barre syndrome
  • AMAN acute motor axonal neuropathy
  • AMSAN acute motor and sensory axonal neuropathy
  • AON acute optic neuritis
  • AD Alzheimer’s disease
  • ALS amyotrophic lateral sclerosis
  • AIE autoimmune encephalitis
  • chronic ataxic neuropathy ophthalmoplegia
  • immunoglobulin M paraprotein immunoglobulin M paraprotein
  • cold agglutinins cold agglutinins
  • disialosyl antibodies CANOMAD
  • chronic meningitis Behcet's disease
  • Central nervous system CNS vasculitis
  • CIDP chronic inflammatory demyelinating polyradiculoneuropathy
  • CLIPPERS chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids
  • GFAP glial fibrillar acidic protein
  • Hashimoto’s encephalitis hypertrophic pachymeningitis, lgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of
  • FIGS. 1A-1B show that MHC II presentation in the CNS centers on a particular MBP region.
  • FIG. 1A demonstrates tire composition of MHC Il-bound peptides derived from proteins labeled as CNS- enriched.
  • FIG. IB shows the exact sequences of peptides bound to MHC II molecules deriving from MBP.
  • FIGS. 2A-2D show that MBP peptides gamer regulatory T cells to suppress CNS autoimmunity.
  • FIG. 2A depicts a schematic for the induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice.
  • EAE experimental autoimmune encephalomyelitis
  • FIG. 2B shows clinical EAE score tracked over 21 days for mice immunized with either MOG35-55, MOG35-55 and MBP33-52, or MOG35-55 and MBP (27-42 subscript); two-way ANOVA with Sidak’s multiple comparison test, p-values, where significant, indicated on plots.
  • FIG. 2C displays representative flow cytometry plots with associated quantifications as in FIG. 2D for evaluation of regulatory T cells in the deep cervical lymph nodes (dCLNs) when comparing between MOG3555 as well as MOG35-55 and MBP2742 co-immunized groups; unpaired two-tailed Student’s t-test, p-values indicated where relevant.
  • dCLNs deep cervical lymph nodes
  • FIGS. 3A-3B show that presentation of MBP peptides is generalizable and similarly suppresses neuroinflammation in a different active EAE model.
  • FIG. 3A depicts CNS-enriched peptides identified across both the brain and dural meninges in SJL/J male mice as well as in fresh frozen human dura.
  • FIG. 3B shows clinical EAE scores tracked over 21 days for SJL/J mice immunized with PLP139-151, PLP139-151 and NEFL160-173, or PLP139-151 and MBP27-42. Statistics done with two-way ANOVA with Sidak’s multiple comparison test, p-values, where significant, indicated on plots..
  • FIG. 4 show s the harnessing of MBP peptides as a therapeutic against CNS autoimmunity.
  • Visualization of EVs by both electron microscopy (EM) and ZetaView 7 nanoparticle tracker visualization.
  • Clinical EAE score tracked over 21 days for mice induced for EAE and with introduction of either empty EVs or MBP27-42 containing EVs via intracistcma magna (i.e.m.) injection prior to onset of disease; two-w ay ANOVA and p-valuc indicated on plot. Peak EAE scores for both conditions also quantified and displayed on the right, unpaired two- tailed Student’s t-test, p-value indicated on plot.
  • FIGS. 5A-5B show 7 analysis of the MHC II peptidome.
  • FIG. 5A depictsepitope mapping by Gibbs cluster analysis of MHC-II-bound peptides of 8-week-old C57BL/6J.I-Ab mice.
  • FIG. 5B depicts distribution for MHC-II-bound peptides enriched for proteins that are expressed at elevated levels in tire CNS. ‘"Other” represents the sum of CNS elevated proteins with only a single MHC-II-bound peptide identified. These include: MAP2, NEFL, DPYSL2, CNP, ISLR2, BCAS1, IRS4, PTGDS, and TBC1D30.
  • FIG. 5A depictsepitope mapping by Gibbs cluster analysis of MHC-II-bound peptides of 8-week-old C57BL/6J.I-Ab mice.
  • FIG. 5B depicts distribution for MHC-II-bound peptides enriched for proteins that are expressed at elevated levels in tire CNS
  • FIG. 6A depicts the experimental scheme for induction of experimental autoimmune encephalomyelitis (EAE).
  • FIGS. 7A shows average clinical EAE score in C57BL6/J mice immunized with MOG35-55 (red), MOG35-55 + MBP12-26 (gray). MOG35-55 + MBP27-42 (blue), or MOG35-55 + MBP2742 (citrullinated) (yellow) on day 0; standard EAE scoring to assess mice daily for 20 days post immunization.
  • FIG. 9A shows average clinical EAE score in SJL/J mice immunized with PLP139-151 (red), PLP139 151 + NF-L160 173 (gray), or PLP139 151 + MBP 2742 (blue) on day 0* standard EAE scoring to assess mice daily for 21 days post immunization.
  • FIG. 9B shows peak EAE score for SJL/J mice as immunized in FIG. 9A.
  • N 5 mice per group, p-values are indicated on the graphs.
  • FIG. 10A shows an immunoblot performed on both supernatant (SN) and pellet post high-speed ultracentrifugation evidencing enrichment of tetraspanins CD9 and CD63, markers of extracellular vesicles, in the pellet.
  • FIG. 10B shows a negative stain by transmission electron microscopy of enriched extracellular vesicles.
  • FIG. 10C shows an experimental scheme (top) depicting tire insertion of MBP27-42 peptides into extracellular vesicles prior to intracistema magna (i.e.m.) injection.
  • FIG. 11A shows average clinical EAE score in C57BL/6J mice immunized with MOG35-55 (red) or CFA only (black) on day 0; standard EAE scoring to assess mice daily for 16 days post immunization.
  • FIG. 11B depicts representative images and
  • FIG. 11C depicts quantification of percent coverage of intracistema magna (i.e.m.) injected OVA647 protein in the dCLN of CFA only or CFA + MOG35-55 immunized mice at peak of EAE disease (day 16). Scale bar, 150 pm. P-values indicated on plots.
  • FIGS. 12A-12C depicts binding motifs for both MHC I alleles H2-Kb (FIG. 12A)and H2-Db (FIG. 12B)as well as the sole MHC II allele H2-Ab (FIG. 12C) in C57BL/6J mice as predicted by both the netMHCpan and netMHCIIpan algorithms.
  • FIGS. 13A-13E show that the CNS MHC II peptidome reveals presentation of endogenous CNS peptides.
  • FIG. 13A depicts a schematic illustrating the mass spectrometric identification of MHC II- bound peptides from brain (including leptomeninges), dural meninges (dura), and lymph nodes, including the deep cervical lymph nodes (dCLNs) and superficial cervical lymph nodes (sCLNs), of C57BL/6J male mice.
  • FIG. 13B shows assessment of the proportion of total unique identified peptides that could be designated as CNS enriched (teal bar); percentages indicated above each individual bar.
  • FIG. 13C depicts a donut plot representation of CNS-enriched, MHC Il-bound peptides identified for each individual tissue. The percent of which is composed by MBP is indicated where relevant.
  • FIG. 13D shows a Venn diagram depicting the relationship between CNS-enriched, MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs.
  • FIG. 13E shows a summary of individual peptide sequences contained within the MBP158-195 region as defined by the MHC II peptidome.
  • FIGS. 14A-14K show that MBP is non-encephalitogenic and fosters immunosuppression.
  • FIG. 14B shows quantification of cell counts (mean ⁇ s.e.m, unpaired two-tailed Student’s t-test).
  • FIG. 14C shows an ELISpot assay performed to measure IL-2 production by CD4+ T cells upon recall with peptides as indicated on the x-axis. Data shown as mean ⁇ s.e.m, two-way ANOVA with Sidak’s multiple comparisons test.
  • FIG. 14E depicts a UMAP visualization of T cells from the draining (inguinal) lymph nodes of MOG35-55 immunized or MOG35-55 and MBP160-175 co-immunized C57BL/6J mice.
  • FIG. 14F shows Log2 fold change determined across the different identified clusters by scRNA-seq when comparing MOG35 55 to MOG35 -55 and MBP160 175 immunizations. Significant differences highlighted on the plot.
  • FIG. 14E depicts a UMAP visualization of T cells from the draining (inguinal) lymph nodes of MOG35-55 immunized or MOG35-55 and MBP160-175 co-immunized C57BL/6J mice.
  • FIG. 14F shows Log2 fold change determined across the different identified clusters by scRNA-seq when comparing MOG35 55 to MOG35 -55 and MBP160 175 immunizations. Significant differences highlighted on the plot.
  • FIG. 14E depicts a
  • FIGS. 15A-15F show that therapeutic delivery of guardian MBP peptides guards against CNS autoimmunity.
  • FIG. 15A depicts a schematic depiction of isolating peptides bound to MHC II molecules in the brain, dura, and spinal cord at the peak (day 16) of MOG35-55 induced EAE.
  • FIG. 15B shows the MHC II peptidomes from EAE and naive mice. Quantified relative abundances for peptides was identified within different antigenic regions to generate a heatmap, illustrating alterations to the repertoire of MHC Il-bound peptides in EAE when compared to naive mice.
  • FIG. 15C depicts a bar graph representation of the relative abundances of MBP peptides contained within the MBP158-195 or MBP196-236 regions for the brain (top) and dura (bottom) when comparing EAE to naive mice.
  • Relative abundances (normalized peak areas) were measured relative to (Cbln 15772) for the brain and (Mbpi96-23e) for the dura.
  • FIG. 15D shows an experimental design depicting i.e.m.
  • FIG. 15F depicts an experimental scheme atop illustrating i.e.m.
  • FIGS. 16A-16H show characterization of the MHC II peptidome in the CNS of C57BL/6J male mice.
  • FIGS. 16A-16D show representative flow cytometry plots depicting the gating strategy used to identify the distribution of MHC II-expressing cells.
  • B cells gated on CD19+ CD11c-
  • dendritic cells DCs, gated on CD19- CD1 lc+
  • macrophages MO, gated on CD19- CD11c-
  • FIG. 16B shows the frequency of the aforementioned antigen presenting cells as a percent of CD45+ MHC 11+ cells for the brain (FIG. 16B), dura (FIG. 16C), dCLNs (FIG. 16D), and sCLNs (FIG. 16E).
  • FIG. 16F shows a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides. The median is represented by a solid line and the first as well as the third quartiles are represented by dashed lines. P-values indicated on plot (one-way ANOVA with Tukey’s multiple comparison test).
  • FIG. 16F shows a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides. The median is represented by a solid line and the first as well as the third quartiles are represented by dashed lines. P-values indicated on plot (one-way ANOVA with Tukey’s multiple comparison test).
  • FIG. 16D shows a Venn diagram depicting the relationship between all unique MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs.
  • FIG. 16H shows a summary of individual peptides identified on the MBP sequence as defined by the MHC II peptidome of C57BL/6J male mice.
  • FIGS. 17A-17F show the characterization of the MHC II peptidome in the CNS of C57BL/6J female mice.
  • FIG. 17A shows a pie chart defining the makeup of CNS-enriched, MHC Il-bound peptides across each individual tissue. Percent of CNS-enriched peptides that are MBP are indicated in the plots, where relevant.
  • FIG. 17B shows a summary of all identified individual peptide sequences derived from MBP in the C57BL/6J female MHC 11 peptidome.
  • FIG. 17C depicts a Venn diagram representation of individual overlapping CNS-enriched peptide sequences identified between C57BL/6J females and males in the brain (including leptomeninges).
  • FIG. 17A shows a pie chart defining the makeup of CNS-enriched, MHC Il-bound peptides across each individual tissue. Percent of CNS-enriched peptides that are MBP are indicated in the plots, where relevant.
  • FIG. 17B shows a summary of
  • FIG. 17D depict a bar graph showing the relative abundance of MBP peptides in the brain that comprised either the MBPiss-iss or MBP196-236 regions in both C57BL/6J males and females. Relative abundances (normalized peak areas) measured against a common identified peptide sequence, Dagl488-531, from which relativity was ascertained.
  • FIG. 17E depicts a Venn diagram representation of individual CNS-enriched overlapping peptide sequences identified between C57BL/6J females and males in the dura.
  • FIG. 17F depicts a bar graph showing the relative abundance of MBP peptides in the dura that comprised either the MBP158-195 or MBP196-236 regions in both C57BL/6J males and females.
  • FIGS. 18A-18F show the characterization of the MHC II peptidome in the CNS of of SJL/J male mice.
  • FIG. 18A depicts a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides in the brain (which also includes the leptomeninges), dura, dCLN, and sCLN of SJL/J mice. The median is represented by a solid line, and the first as well as the third quartiles are represented by dashed lines.
  • FIG. 18B shows evaluation of the proportion of total unique identified peptides designated as CNS enriched (teal bar) in the brain, dura, dCLN, and sCLN; percentages indicated above each individual bar.
  • FIG. 18C shows evaluation of the proportion of total unique identified peptides designated as CNS enriched (teal bar) in the brain, dura, dCLN, and sCLN; percentages indicated above each individual bar.
  • FIG. 18C shows evaluation
  • FIG. 18E depicts a donut plot representation of all CNS-enriched peptides identified in the MHC II peptidome of SJL/J male mice for the brain, dura, and sCLN. The part of the whole for which MBP represents is indicated in the plot, where relevant.
  • FIG. 18F shows a depiction of all individual peptide sequences deriving from MBP identified in the SJL/J male MHC II peptidome.
  • FIGS. 19A-19E show that endogenous guardian MBP peptides protect across different models of neuroinflammation.
  • FIG. 19A depicts an experimental design of immunizing either C57BL/6J or SJL/J mice with MOG35-55 or PLP139-151 peptides, respectively, with or without MBP or NEFL peptides to actively induce EAE. Separately, C57BL/6J mice were also immunized individually with myelin associated peptides to perform ELISpot assay.
  • Plots display mean ⁇ s.e.m and represent two independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test.
  • Plots display mean ⁇ s.e.m and represent three independent experiments.
  • Plots display mean ⁇ s.e.m and represent two independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test.
  • FIGS. 20A-20E show that peripheral presentation of MBP peptides induces conventional Tregs.
  • FIG. 20A depicts a dot plot of population markers from single cell RNA-sequencing scaled by percentage of cells expressing marker genes for each T cell cluster.
  • FIG. 20B shows a representative gating strategy for flow 7 cytometry used to define within CD4+ T cells, CTLA-4+ Foxp3- suppressor T cells as well as conventional Foxp3+ regulatory T cells (Treg). CTLA-4+ Foxp3- CD4+ T cells further gated to assess CD39.
  • PD-1 CD279
  • IL-10 expression FIGS. 20C-20D show representative flow cytometry plots with relevant quantifications (FIG.
  • FIGS. 21A-21G show 7 that neuroinflammation alters the MHC Il-bound repertoire of autoantigens.
  • FIG. 21C show s a pie chart depiction of CNS-enriched, MHC Il-bound peptides identified in the brain and spinal cord at the peak of EAE disease. Below is a visual summary of individual MBP sequences identified.
  • FIG. 21D shows a population pyramid representation of the distribution of amino acids identified at the C -terminus as a percent of the total MHC Il-bound peptides, indicating cleavage preference between EAE and naive mice.
  • FIG. 21E shows UMAP projections of the Jordao ct al. dataset displaying broad cell lineages, color-coded accordingly, when analyzing naive, pre-symptomatic, and EAE mice.
  • eDCs conventional dendritic cells.
  • migDCs migratory dendritic cells.
  • FIG. 21F shows a volcano plot depicting differences in gene expression when comparing microglia in EAE-induced mice to controls. Arrows indicate peptidases that could be identified.
  • FIG. 21G shows gene ontologies found to be significantly upregulated in microglia when comparing between EAE and naive mice.
  • FIGS. 22A-22F show that delivery of encapsulated MBP peptides directly into the CSF induces unconventional suppressor CD4+ T cells.
  • FIG. 22A shows an immunoblot performed on the supernatant (SN) and resuspended pellet after ultracentrifugation evidencing enrichment of tetraspanins, CD9 and CD63, markers of extracellular vesicles (EVs). Below 7 is a negative stain by transmission electron microscopy depicting enriched EVs, scale bar indicated on the panel.
  • FIG. 22B shows that gating strategy for flow cytometry analysis to define within CD4+ T cells, CTLA-4+ Foxp3- unconventional suppressor T cells as well as conventional Foxp3+ Treg.
  • FIGS. 22C-22E show quantification of unconventional CTLA-4+ Foxp3- suppressor T cells as well as conventional Foxp3+ Treg as a frequency of CD4+ T cells of the deep cervical lymph nodes (FIG. 22C), superficial cervical lymph nodes (FIG. 22D), and spleen (FIG. 22E).
  • FIGS. 23A-23G show candidates for regulatory peptides against CNS autoimmunity.
  • Clinical EAE scores tracked over 19 days post immunization with MOG35-55 or MOG35-55 and Tubb3 peptide 1 (FIG. 23A); MOG35-55 or MOG35-55 and Tubb3 peptide 2 (FIG. 23B); MOG35-55 or MOG35-55 and Tubb3 peptide 3 (FIG. 23C); MOG35.55 or MOG35-55 and Tubb3 peptide 4 (FIG. 23D); MOG35-55 or MOG35-55 and Map2 (FIG. 23E); MOG35-55 or MOG35-55 and Neftn peptide 1 (FIG.
  • autoreactive T cells are well-known for provoking autoimmune disease, their protective roles in limiting tissue damage have been described in heart, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer’s disease, stroke, and traumatic injury, among others. With evidence supporting a suppressive and beneficial function for autoreactive T cells, the molecular and cellular mechanisms interconnecting the CNS and the peripheral immune system requires further investigation.
  • Tire compositions and methods of the present disclosure are based on the surprising discovery that in an experimental MOG35-55 -induced autoimmune encephalomyelitis (EAE) model, co-immunization of MOG35-55 with MBP33-42 peptides ameliorated EAE disease. Further analyses by flow cytometry prior to the peak of EAE disease unveiled an elevated frequency of regulatory Foxp3-expressing CD4+ T cells in CNS-associated tissues of mice coimmunized with the MBP peptides.
  • EAE autoimmune encephalomyelitis
  • CSF tire cerebrospinal fluid
  • MBP3342 fragment is equivalent to the MBP166-175 fragment, when accounting for Goli numbering differences.
  • Tire CNS antigen repertoire used to develop the compositions and methods of the present disclosure overcome the shortcomings of existing methods that have hampered our capacities to efficaciously employ antigen-specific immune -tolerizing therapeutics.
  • Inadequate knowledge of the repertoire of antigens presented during homeostasis of the CNS is one barrier overcome by utilizing an optimized technique to unravel native peptides bound to major histocompatibility complex class II (MHC II) molecules in the CNS and its borders.
  • MHC II major histocompatibility complex class II
  • the present disclosure extensively characterizes the MHC II peptidome in CNS during homeostasis, providing the ability to broadly understand the specificities of autoreactive T cells in the CNS.
  • the identified peptides described herein are useful in directing the development of MHC II tetramers, allowing for tracking and the development of phenotype autoreactive CNS-specific T cells.
  • the methods described herein provide insights into the function of relevant antigen presentation in the CNS to harbor local populations of autoreactive T cells - an understanding of which illuminates mechanisms the CNS employs to acquire immune privilege.
  • Another critical barrier to the development of antigen-specific immunotherapies derives from an insufficient understanding of disease-initiating autoantigens and subsequent epitope spreading often arising as a consequence of chronic autoimmune or inflammatory response.
  • the present disclosure overcomes this barrier by leveraging an established platform to define the MHC II peptidome to understand how the repertoire of autoantigens presented on MHC molecules diverges from homeostasis with neuroinflammation.
  • existing antigen-specific therapeutic strategies also face a barrier of being constrained to identified pathogenic epitopes and finding ways to manipulate either their sequence or their delivery to drive tolerogenic immune responses.
  • the methods disclosed herein circumvent this barrier by taking advantage of the knowledge of CNS-derived endogenous antigen presentation in homeostasis to inform antigen-specific immunotherapies.
  • the disclosed methods therapeutically deliver a homeostatic, CNS-specific, MHC Il-bound autoantigen to suppress and protect against CNS autoimmunity.
  • compositions and methods described herein provide an expanded comprehension of the repertoire of autoantigens presented and of autoreactive T cells within the CNS, thus furthering understanding of the mechanisms supporting CNS immune privilege and immune tolerance by extension.
  • Tire methods disclosed herein foster an understanding that will guide antigen-specific immunotherapies, enabling the induction of immune tolerance in other tissues to protect against allo- or autoimmune attack for clinical benefit.
  • the term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ⁇ 5%, but can also be ⁇ 4%, 3%. 2%,1%, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
  • the term “subject” refers to a mammal, preferably a human.
  • the mammals include, but are not limited to, humans, primates, livestock, rodents, and pets.
  • a subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.
  • control population refers to a subject, or group of subjects, who are clinically determined to not have a disease.
  • the medical care may be a diagnostic test, a therapeutic treatment, and/or a prophylactic or preventative measure.
  • the object of therapeutic and prophylactic treatments is to prevent or slow down (lessen) an undesired physiological change or disease/disorder.
  • Beneficial or desired clinical results of therapeutic or prophylactic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder orthose in which the disease, condition or disorder is to be prevented. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of disease.
  • heterologous DNA sequence refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, tire use of DNA shuffling or cloning.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
  • Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
  • a '‘promoter” is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid.
  • An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus.
  • a promoter can include necessary nucleic acid sequences near tire start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a "transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit the translation of a specific RNA molecule of interest.
  • compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
  • the '‘transcription start site” or "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein-encoding sequences in tire 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
  • operably-linked refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • the two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent.
  • a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
  • a "construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
  • a construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule.
  • constructs can include but are not limited to additional regulatory nucleic acid molecules from, e g., the 3 '-untranslated region (3' UTR).
  • Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct.
  • These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
  • Tire term "transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in a genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
  • Transformed refers to a host cell or organism such as a bacterium, cyanobacterium, animal, or plant into which a heterologous nucleic acid molecule has been introduced.
  • Tire nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
  • the term “untransfonned” refers to normal cells that have not been through the transformation process.
  • Wild-type refers to a virus or organism found in nature without any known mutation.
  • Nucleotide and/or amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Mcgalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • percent sequence identity X/Y100, where X is tire number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B. the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by He, Leu by He, and Ser by Thr.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium- containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium- containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids.
  • An amino acid sequence can be modulated with the help of art- known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
  • Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed.. Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
  • transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Exemplary nucleic acids which may be introduced to a host cell include, for example. DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species but are incorporated into recipient cells by genetic engineering methods.
  • exogenous is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in tire transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express.
  • tire term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell.
  • Tire type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
  • Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
  • RNA interference e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA)
  • siRNA small interfering RNAs
  • shRNA short hairpin RNA
  • miRNA micro RNAs
  • RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen).
  • sources e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen.
  • siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Ccnix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinfomiatics & Research Computing).
  • Traits influential in defining optimal siRNA sequences include G/C content at the termini of tire siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.
  • compositions which comprise a peptide as described herein, or a nucleic acid encoding the peptide (e.g., an rnRNA or a vector encoding the peptide).
  • Tire composition may be a phannaceutical composition, which comprises the peptide or nucleic acid, and one or more phannaceutically acceptable excipients.
  • peptides which may be used in a composition or a method, or for informing the encoding sequence of a nucleic acid.
  • Such peptides are based on those from a discovered repertoire of CNS-derived endogenous regulatory peptides (e.g., guardian peptides), which bind to major histocompatibility complex (MHC) molecules (e.g., MHC class I and MHC class II) where they serve as autoantigens important in autoreactive T cell recognition.
  • MHC guardian peptides are presented throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes, where they function in the protection against autoimmunity.
  • guardian peptides are diminished in certain disease states, such as neuroinflammatory disease.
  • Administration of a peptide as described herein e.g., a peptide derived from an MHC guardian peptide
  • a subject mediates autoimmunity (e.g., CNS autoimmunity) by supplementing the function of MHC guardian peptides in immune modulation.
  • the peptide may be used for enhancement or expansion of suppressor CD4+ T cell population (e.g., for reduction of CNS autoimmune disease)
  • the peptide may be derived from an MHC guardian protein that is an MHC I guardian peptide, MHC II guardian peptide, or both.
  • the peptide may be derived from an MHC guardian protein that is CNS-enriched, or an MHC guardian protein that is not CNS-enriched.
  • CNS-enrichment may be elevated expression of the protein in the CNS relative to a control tissue type. In some embodiments, the elevated expression is at least about 2-fold, at least about 3 -fold, at least about 4-fold, or at least about 5 -fold relative to a control.
  • the peptide may be derived from an MHC guardian protein that is a human MHC guardian protein, a murine (e.g., mouse) guardian protein, or both.
  • the peptide comprises a fragment of a protein, wherein the fragment of the protein binds to an MHC molecule, hr some embodiments, the protein is expressed at elevated levels in the CNS. In some embodiments, the protein is CNS enriched. In some embodiments, the protein is not expressed at elevated levels in the central nervous system (CNS), or is expressed at decreased levels in the CNS. In some embodiments, the protein is not enriched in the CNS. In some embodiments, the peptide is a fragment of a protein selected from TABLE 4.
  • a peptide as described herein is an MHC guardian peptide. In some embodiments, the peptide binds to MHC I, MHC II, or to both. [085] In some embodiments, a peptide comprises or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof.
  • a modified sequence may comprise (e.g., relative to a wild-type MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619), one or more of: oxidation of methionine; deamidation of glutamine; deamidation of asparagine; citrullination of arginine to citrulline; oxidation of cysteine to cysteic acid; or pyroglutamic acid formation from glutamine.
  • the peptide does not have oxidation of methionine.
  • the peptide does not have deamidation of glutamine.
  • the peptide does not have deamidation of asparagine.
  • the peptide does not have citrullination of arginine to citrulline. In some embodiments, the peptide does not have oxidation of cysteine to cysteic acid. In some embodiments, the peptide does not have pyroglutamic acid formation from glutamine.
  • a modified sequence may also be a variant sequence (e.g., relative to a wild-type derived MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619).
  • a variant sequence retains its ability to bind to MHC (e.g., MHC I or MHC II).
  • a variant sequence is at least 80%, at least 85%, at least 90%, or at least 95% identical to a wild-type MHC guardian peptide sequence or to any one of SEQ ID NOS: 1-14619.
  • a variant sequence may comprise one or more amino acid additions or deletions.
  • a variant sequence may comprise one or more conservative amino acid substitutions.
  • the conservative amino acid substitution is based on a side chain characteristic at the one or more modified positions. In some embodiments, the conservative amino acid substitution is a substitution set forth in TABLE 1 or TABLE 2. In some embodiments, the conservative amino acid substitution is based on the amino acid identity at the one or more modified positions. In some embodiments, the conservative amino acid substitution is a substitution set forth in TABLE 3.
  • a modified sequence may also be a conjugated sequence (e.g., a wild-type derived MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619), wherein the peptide is conjugated to a moiety.
  • the moiety may be for enhancing delivery or enhancing targeting of the peptide to the CNS.
  • TABLES 5-10 categorize the peptides of SEQ ID NOS: 1-14619 based on their CNS enrichment and presence in the MHC peptidome of humans, mouse, or both. This information, along with the protein and start/stop positions from which the peptides derive from, are provided in the remarks of each sequence in the sequence listing accompanying this patent.
  • a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 5, or a modified sequence thereof.
  • a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 6, or a modified sequence thereof.
  • a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 8, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 9, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 10, or a modified sequence thereof.
  • the peptide is a fragment of a myelin basic protein (MBP).
  • the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785. 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782. 5783, 5784, 5785, 5786, 5787, 5788, 5789. 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754.
  • the peptide is a fragment of a tubulin beta 3 class III (TUBB3) protein.
  • the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: IN 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, and 14155, or a modified sequence thereof.
  • the peptide is a fragment of a neurofilament medium polypeptide (NEFM).
  • the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:295, 11267, 11268, 11269. 11270, 11271, 11272, 11273, 11274, 11275, 11276, 11277, 11278. 11279, 11280, 11281, 11282. 11283, 11284, and a modified sequence thereof.
  • the peptide comprises or consists of the amino acid sequence of, FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
  • nucleic acids encoding a peptide as described herein, or a modified sequence thereof.
  • a nucleic acid encodes a fragment of a protein that binds to MHC (e.g., MHC I or MHC II).
  • a nucleic acid encodes an MHC guardian peptide.
  • a nucleic acid encodes an MHC I guardian peptide, an MHC II guardian peptide, or an MHC I/II guardian peptide (i.e., both).
  • a nucleic acid may encode for a peptide comprising an MHC -binding fragment of a protein set forth in TABLE 4.
  • a nucleic acid may encode for a peptide comprising or consisting of an amino acid sequence set forth in any one of the SEQ ID NOS set forth in TABLE 5, 6, 7, 8, 9, or 10.
  • a nucleic acid may encode for a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof.
  • a nucleic acid encodes a peptide comprising an MHC-binding fragment of a myelin basic protein (MBP).
  • the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438. 5439, 5773, 5774, 5775, 5777, 5778, 5779. 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788. 5789.
  • a nucleic acid encodes a peptide comprising an MHC -binding fragment of a tubulin beta 3 class III (TUBB3) protein.
  • the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: IN 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, or 14155, or a modified sequence thereof
  • a nucleic acid encodes a peptide comprising an MHC-binding fragment of a neurofilament medium polypeptide (NEFM).
  • the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS:295, 11267, 11268, 11269, 11270, 11271, 11272, 11273. 11274, 11275, 11276, 11277, 11278, 11279, 11280, 11281, 11282, 11283. 11284, or a modified sequence thereof.
  • the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence of: FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
  • vectors comprising a nucleic acid encoding a peptide as described herein.
  • There may be one or more vectors, wherein each of the one or more vectors comprises at least one or more nucleic acids, as described herein.
  • a vector or multiple vectors may encode multiple different peptides. Such vectors enable production of a transcribable nucleic acid molecule that encodes a peptide as described herein.
  • a vector may further comprise a transcription initiation site, and/or a promoter operably-linked to a transcribable nucleic acid molecule encoding a peptide described herein.
  • a vector may be a non-viral vector.
  • the non-viral vector is a physical vector (e.g., electroporation, sonoporation, magnetofection: or hy droporation).
  • the non-viral vector is a chemical vector.
  • the chemical vector is an inorganic particle based, lipid based (e.g., solid or liquid), polymer based, or polypeptide based.
  • the inorganic particle based vector is selected from calcium phosphate, silica, gold, or magnetic nanoparticle.
  • the nucleic vector is a nucleic acid/cationic lipid (lipoplex), nucleic acid/cationic polymer (polyplex), or nucleic acid/cationic polymer/cationic lipid (lipopolyplex).
  • the vector is selected from a cationic lipid, lipid nano emulsion, or solid lipid nanoparticle.
  • the vector is a polypeptide based vector, a polymer based vector, or a synthetic vector.
  • the synthetic vector is selected from a polyethylene imine (PEI), chitosal, poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA), dendrimer, polyphosphoester, or polymethacrylate .
  • PEI polyethylene imine
  • PLA poly (DL- Lactide)
  • PLGA Poly ( DL-Lactide- co- glycoside)
  • dendrimer polyphosphoester
  • polymethacrylate polymethacrylate
  • a vector may be a non-viral vector.
  • the non-viral vector is a physical vector (e.g., electroporation, sonoporation, magnetofection; or hy droporation).
  • the non-viral vector is a chemical vector.
  • the chemical vector is an inorganic particle based, lipid based (e.g., solid or liquid), polymer based, or polypeptide based.
  • the inorganic particle based vector is selected from calcium phosphate, silica, gold, or magnetic nanoparticle.
  • the nucleic vector is a nucleic acid/cationic lipid (lipoplex), nucleic acid/cationic polymer (polyplex), or nucleic acid/cationic polymer/cationic lipid (lipopolyplex).
  • the vector is selected from a cationic lipid, lipid nano emulsion, or solid lipid nanoparticle.
  • the vector is a polypeptide based vector, a polymer based vector, or a synthetic vector.
  • the synthetic vector is selected from a polyethylene imine (PEI), chitosal. poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA). dendrimer, polyphosphoester, or poly ethacrylate.
  • the nucleic acid may comprise a mRNA encoding a peptide disclosed herein.
  • a nucleic acid as described herein may also be encapsulated.
  • a nucleic is encapsulated by a lipid nanoparticle (LNP), polyplex, polymeric nanoparticle, lipopolyplex (LPP), or cationic polypeptide.
  • LNP lipid nanoparticle
  • a nucleic acid may be encapsulated by any particle that is phagocytosed (e.g., for delivery ).
  • a nucleic acid may be encapsulated by any particle that can be up taken by antigen presenting cells.
  • a vector may a viral vector.
  • the viral vectors may? be derived from a virus having CNS or brain tissue specificity (e.g. tropism).
  • the viral vector is derived from a virus having CNS or brain tissue specificity and not having liver, heart, and/or muscle tropism.
  • the viral vector may be derived from an adenovirus, adeno-associated virus, or retrovirus.
  • the retrovirus is a lentivirus.
  • host cells comprising a nucleic (e.g., a vector or construct) encoding a peptide as described herein.
  • Tire host cells may be used for expression (e.g., production) of a peptide as described herein, or for maintaining a nucleic (e.g., vector or construct) as described herein.
  • the host cell may or may not be transformed (e.g., transgenic
  • compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example. Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such fonnulations will contain a therapeutically effective amount of a peptide as described herein, or a nucleic acid encoding the peptide, which can be in purified form, together with a suitable amount of one or more pharmaceutically acceptable carriers or excipients so as to provide the form for proper administration to the subject.
  • formulation refers to preparing a drug in a form suitable for administration to a subject, such as a human.
  • a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
  • Tire term "pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Generally, they include substances that are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF. etc. may also be used.
  • compositions can include a solvent, dispersion media, coating, antibacterial and antifungal agent, isotonic, or absorption-delaying agent.
  • a solvent dispersion media
  • coating antibacterial and antifungal agent
  • isotonic or absorption-delaying agent.
  • absorption-delaying agent a solvent, dispersion media, coating, antibacterial and antifungal agent, isotonic, or absorption-delaying agent.
  • the use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed ), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • a “stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
  • Tire formulation should suit the mode of administration.
  • the agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to. parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
  • the individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents.
  • Such biologically active or inert agents may be in fluid or mechanical communication w itli the agent(s) or attached to tire agent(s) by ionic, covalent. Van der Waals, hydrophobic, hydrophilic, or other physical forces.
  • Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e.g.. change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • inducers e.g. change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below.
  • therapies described herein one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.
  • the peptide may in a composition (e.g., a pharmaceutical composition) as described herein, which is administered to the subject.
  • the neuroinflammation-related disorder or autoimmune neural disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), acute optic neuritis (AON).
  • ADAM acute disseminated encephalomyelitis
  • AIDP acute inflammatory demyelinating polyradiculoneuropathy
  • GBS Guillain-Barre syndrome
  • AMAN acute motor axonal neuropathy
  • AMSAN acute motor and sensory axonal neuropathy
  • AON acute optic neuritis
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • AIE autoimmune encephalitis
  • chronic ataxic neuropathy ophthalmoplegia
  • immunoglobulin M paraprotein immunoglobulin M paraprotein
  • cold agglutinins cold agglutinins
  • disialosyl antibodies CANOMAD
  • chronic meningitis Behcet's disease
  • Central nervous system CNS vasculitis
  • CIDP chronic inflammatory demyelinating polyradiculoneuropathy
  • CLIPPERS chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids
  • GFAP glial fibrillar acidic protein
  • Hashimoto’s encephalitis hypertrophic pachymeningitis, IgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of un
  • the acute CNS injury' is selected from a spinal cory injury, traumatic brain injury’, spinal cord injury’, optic nerve injury, and stroke.
  • Methods described herein are generally performed on a subject in need thereof.
  • a subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neuroinflammation-related disorder, autoimmune neural disorder, or an acute CNS injury.
  • a determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art.
  • the subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens.
  • the subject can be a human subject.
  • a safe and effective amount of the peptide is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.
  • an effective amount of the peptide (e.g., MBP peptide) described herein can substantially inhibit a neuroinflammation-related disorder or autoimmune neural disorder, slow the progress of a neuroinflammation-related disorder or autoimmune neural disorder, or limit the development of a neuroinflammation-related disorder or autoimmune neural disorder.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • the disclosed composition is infused, injected, or otherwise introduced into the cerebrospinal fluid (CSF) of the subject.
  • CSF cerebrospinal fluid
  • the peptide e.g., MBP peptide
  • the extracellular vesicles may be derived from dendritic cells.
  • a therapeutically effective amount of the peptide e.g., MBP peptide
  • the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to prevent, reduce, or reverse a neuroinflammation-related disorder or autoimmune neural disorder.
  • compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
  • Toxicity and therapeutic efficacy of compositions described herein can be detennined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al.
  • treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof.
  • treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms.
  • a benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
  • Administration of the peptide can occur as a single event or over a time course of treatment.
  • meningeal immune cells can be administered daily, weekly, bi-weekly, or monthly.
  • the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
  • Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for prevention, reduction, or reversal of the neuroinflammation-related disorder or autoimmune neural disorder.
  • the peptide can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.
  • the peptide can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory.
  • Simultaneous administration can occur through the administration of separate compositions, each containing one or more of peptide, an antibiotic, an anti-inflammatory, or another agent.
  • Simultaneous administration can occur through the administration of one composition containing two or more peptides, an antibiotic, an anti-inflammatory, or another agent.
  • the peptides e g., MBP peptides
  • the peptide can be administered before or after tire administration of an antibiotic, an anti-inflammatory, or another agent.
  • Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art.
  • the agents and composition can be used therapeutically either as exogenous materials or as endogenous materials.
  • Exogenous agents are those produced or manufactured outside of the body and administered to the body.
  • Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
  • Agents and compositions described herein can be administered in a variety of methods well- known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery' of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
  • Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors.
  • an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site.
  • polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof.
  • a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
  • Agents can be encapsulated and administered in a variety of carrier delivery systems.
  • carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331).
  • Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve tire transport of tire drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product.
  • Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
  • Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups.
  • the candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • a candidate molecule can be a compound in a library database of compounds.
  • One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chern Inf Model 45, 177-182).
  • Candidate molecules for screening according to the methods described herein include both lead- like compounds and drug-like compounds.
  • a lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948).
  • a drug -like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g.. less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Phann. Tox. Methods 44, 235-249).
  • Initial screening can be performed with lead-like compounds.
  • kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein.
  • the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to compositions containing a peptide as described herein (e.g., MBP peptide) optionally enclosed in extracellular vesicles as described herein.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, and sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil- lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, and the like.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic -readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.
  • compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed.. Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988.
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • Example 1 Investigating CNS immune privilege - autoantigens fend against CNS autoimmunity
  • MHC major histocompatibility complex
  • autoreactive T cells are well-known for provoking autoimmune disease, their protective roles in limiting tissue damage have been described in heart, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer’s disease, stroke, traumatic injury, among others.
  • CNS central nervous system
  • the molecular and cellular mechanisms interconnecting the CNS and the peripheral immune system requires further investigation. This search has benefited from the recent rediscovery of a bona fide lymphatic network in the meninges, the membranous coverings that envelop the CNS.
  • the brain meninges provide a landscape of opportunities to examine the local presentation of autoantigens to autoreactive T cells.
  • meningeal lymphatic vessels serve as conduits for the drainage of antigens and cells to secondary lymphoid organs, specifically the deep cervical lymph nodes (dCLNs).
  • dCLNs deep cervical lymph nodes
  • the preliminary' data utilized a specialized platform for immunopeptidomics, spearheading an understanding of the CNS MHC II peptidome.
  • MBP myelin basic protein
  • an abundance of peptides derived from myelin basic protein (MBP) were identified that centered around a common sequence, MBP33-42, was identified.
  • EAE experimental autoimmune encephalomyelitis
  • co-immunization of MOG35-55 with our identified MBP peptides ameliorated EAE disease.
  • Further analyses by flow cytometry prior to the peak of EAE disease unveiled an elevated frequency of regulatory Foxp3 -expressing CD4+ T cells in CNS-associated tissues of mice co-immunized with MBP peptides.
  • Aim 1 is to address a hypothesis that CNS derived endogenous peptides promote differentiation of regulatory T cells, and thus, modulate CNS autoimmunity. Inducing EAE by co-immunizing MOG with MBP peptides resulted in increased frequencies of regulatory T cells. It was hypothesized that MBP specific T cells within the CNS possess regulatory' features that contribute to CNS immune tolerance. To bolster the idea that MBP peptides cultivate Foxp3+ regulatory T cells to ameliorate disease, EAE is induced with MOG and MBP peptides in Foxp3DTR mice with or without diphtheria toxin. Furthermore, the specificity of Foxp3+ regulatory T cells for MBP peptides in the meninges and dCLN is examined utilizing MBP-specific MHC II tetramers.
  • Aim 2 is to evaluate a hypothesis that neuropathology limits the presentation of homeostatic epitopes on MHC II molecules. Tissue damage and inflammation lead to epitope spreading, which is thought to further contribute to autoimmune pathology. However, it remains elusive whether the presentation of homeostatic epitopes, such as MBP, are also affected by inflammation. It was expected that neuropathology limits the presentation of MBP peptides, especially those containing the MBP33-42 sequence. MHC II peptidomics of the brain meninges and draining dCLNs was performed at the peak of EAE disease.
  • Aim 3 is to test a hypothesis that delivery of CNS derived endogenous peptides broadly safeguards against neuroinflammation -induced pathology. Introducing MBP-containing vesicles into the CSF could suppress EAE disease. Moreover, co-immunization of our MBP peptides with my'elin proteolipid protein (PLP)i39-i5i mitigated EAE disease in SJL/I mice. Thus, it was hypothesized that MBP peptides can be implemented therapeutically to broadly limit neuroinflammation and restore immune tolerance. To demonstrate this, MBP containing EVs were delivered at different time points after the onset of EAE disease.
  • EAE was actively induced in different strains, including C57BL/6J, SJL/J, and BIO.
  • PL mice while delivering MBP-containing vesicles into the CSF to demonstrate broad treatment efficacy of our identified MBP peptides.
  • our identified peptides would help direct the development of MHC II tetramers, allowing the tracking and phenotyping of autoreactive CNS-specific T cells. This provides insight into the function of relevant antigen presentation in the CNS to harbor local populations of autoreactive T cells - an understanding of which elucidates mechanisms the CNS employs to acquire immune privilege. Aim Isought to address these two major barriers.
  • Another critical barrier to the development of antigen-specific immunotherapies derives from an insufficient understanding of disease-initiating autoantigens and subsequent epitope spreading often arising as a consequence of chronic autoimmune or inflammatory response 1 ’ 5 .
  • These studies overcome this barrier by leveraging our platform technology to define the MHC II peptidome to understand how the repertoire of autoantigens presented on MHC molecules diverges from homeostasis with neuroinflammation.
  • Aim 2 provides a strategy to address this.
  • antigen-specific therapeutic strategies also face a critical barrier of being constrained to identified pathogenic epitopes and finding ways to manipulate either their sequence or their delivery to drive tolerogenic immune responses 6 ’ 10 .
  • autoimmune diseases remain a major public health concern, incapacitating many individuals with notably increasing rates, ranging from 3-6% annually 11 12 . In fact, 4.5% of individuals w orldw ide and more than 24 million people in the United States alone are afflicted by a variety of autoimmune disorders 13 14 . Moreover, with enhanced risks for developing another autoimmune condition 15 16 , cancers 17 , and psychiatric disorders 18-21 , autoimmune diseases negatively impact an individual's quality of life, often leading to lifelong disabilities with undeniable socioeconomic burden 22,23 .
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • antigen-specific immunotherapies offer an appealing alternative to precisely target organ-specific autoimmunity- and has shown promise in different autoimmune conditions 37-41 .
  • antigen-specific approaches have not always been effective and in some cases have been detrimental 42-45 , exemplifying an incomplete mechanistic understanding of autoantigen presentation in maintaining immune tolerance.
  • MHC molecules readout the state of the cell and the physiology of the tissue at large through shaping the repertoire of antigens presented.
  • antigens were thought to predominantly derive from endogenous sources for MHC I and exogenous sources for MHC II molecules, in recent years, this distinct separation for peptide sources has been blurred.
  • Exogenously derived antigens have been described to funnel through different intracellular pathways to present on MHC I molecules - a process termed cross-presentation 52-55 .
  • cross-presentation 52-55 On the other hand, it is also becoming more and more appreciated the abundance of endogenously derived peptides presented on MHC II molecules through which autophagy is thought to play a contributing role 56-58 .
  • Aim 1 builds upon preliminary findings to elucidate regulatory mechanisms of autoantigen dependent protection against autoimmunity.
  • Aim 2 contextualizes the repertoire of autoantigens presented on MHC II molecules in physiology and pathology.
  • Aim 3 advances the therapeutic delivery of autoantigens to reinforce immune tolerance, effectively defending against autoimmune pathology.
  • MBP-derived peptides were prominently presented in tire brain meninges, comprising 77% of peptides that could be defined as CNS elevated (FIG. 5B).
  • the abundance of MBP presentation in the brain meninges was surprising and elucidation of its role in CNS immune tolerance was sought.
  • MBP33-42 a vast majority of MBP-derived peptides centered around a common amino acid sequence, MBP33-42.
  • MBP peptides that were identified in our MHC II peptidome were synthesized, which either included or excluded the common MBP33-42 sequence.
  • Endogenous peptide suppresses myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)
  • MBP2742 regulated the pathogenicity of MOG ⁇ ⁇ -induccd EAE by modulating the expression or functionality of RORyt.
  • 8-week-old C57BL/6J mice were immunized with either MOG35-55 or MOG35-55 + MBP27-42 to induce EAE.
  • dCLNs were harvested at day 13 and the brain meninges at day 16 post EAE induction and flow cytometry was perfonned to assess for any changes to T cells.
  • MHC II peptidome analysis of the brain meninges of SJL/J mice revealed an abundant presentation of MBP peptides that similarly contained the MBP33-42 sequence.
  • MBP27-42-mediated protection against neuroinflammation could be generalizable, we utilized another active EAE model with SJL/J mice.
  • PLPjug 151 myelin proteolipid protein
  • NF-L neurofilament light polypeptide
  • NF-L160-173 an endogenous peptide of similar length to MBP27-42, was selected as an appropriate control peptide with evidence supporting its presentation on MHC II molecules in EAE with limited T cell reactivity in SJL/J micegs.ge. Moreover, NF-L160-173 was identified as an MHC II -bound peptide in the brain meninges of SJL/J mice. Consistent with literature, immunization of PLP139-151 in young male SJL/J mice exhibited monophasic EAE disease 97 . To our surprise, co-immunization with MBP2742 but not NF-Li 60 -i73 demonstrated significant suppression of EAE with a notable downward trend to peak EAE scores (FIGS.
  • mice in the experiments were used in the experiments. If mice in the experimental group were obtained from an outside provider, so were the respective controls. Transgenic mice and their wild-type littermate controls were bred in the same facility. Assessment was made using both males and females.
  • Aim 1 CNS derived endogenous peptides promotes differentiation of regulatory T cells, and modulate CNS autoimmunity
  • EAE enhanced green fluorescent protein
  • mice 8- yveek-old Foxp3EGFP mice are utilized, yvhich only express enhanced green fluorescent protein (EGFP) under the control of the Foxp3 promoter.
  • EAE is induced in both transgenic mice with either MOG35-55 alone or MOG35 55 + MBP27-42. Diphtheria toxin is injected intraperitoneally and intravenously (200 ng) on day 3 post EAE onset. All experimental groups are subjected to standard EAE scoring for up to 21 days. Tissues are harvested, including brain meninges, at day 16, the peak of EAE disease, for analysis by both floyv cytometry and immunohistochemistry.
  • Foxp3EGFP control mice recapitulate of preliminary findings is expected, demonstrating an upward trend to Foxp3 expression in the brain meninges following co-immunization of MOG35-55 + MBP27-42 when compared to MOG35-55.
  • Specific ablation of Foxp3+ regulatory T cells in Foxp3DTR mice is expected to abrogate MBP2742 dependent amelioration ofEAE, exhibiting a mechanism that relies on Foxp3 -mediated suppression.
  • MBP2742 is presented on MHC class II molecules in the CNS during homeostasis and that MBP27-42 affords protection against MOG3555 -induced EAE.
  • MHC II tetramers were recently developed using the shared amino acid sequence, MBP3342. These tetramers are tested and calibrated using lymphocytes isolated from the C57BL/6J mice immunized with MBP27-42 peptides boosted with complete Freund’s adjuvant (CFA) bilaterally in flank regions. One week is allowed for generation of a sufficient immune response prior to isolation of the draining inguinal lymph nodes.
  • CFA complete Freund’s adjuvant
  • T cell hybridoma clones In tandem, the isolated T cells are fused with BW5147, a thymic lymphoma cell line, to generate T cell hybridoma clones.
  • In vitro antigen presentation assays are performed by co-culturing individual T cell clones with M12.C3, a B lymphoma cell line serving as the antigen presenting cell, in the presence of MBP2742 Using IL-2 enzyme linked immunosorbent assay (ELISA) as our readout for T cell activation, T cell clones specific to MBP2742 are selected. With guidance by MHC II binding affinity algorithms, key amino acids of MBP2742 are mutated to identify residues that abrogate activation of MBP2742 specific T cell clones.
  • ELISA enzyme linked immunosorbent assay
  • MBP27-42 specific MHC II tetramers By discerning critical recognition events for peptide-MHC II and T cell receptor interaction, the development of MBP27-42 specific MHC II tetramers can be better directed.
  • MBP2742 specific T cells are phenotyped in the brain meninges and dCLN by flow cytometry of wild-type mice, mice immunized with MBP2742 alone, and mice co-immunized with MOG35-55 + MBP2742. Cell surface markers and transcription factors, including Foxp3, are stained. In doing so, whether the CNS harbors a population of MBP27-42 specific CD4+ T cells that express markers of regulation, such as Foxp3, contributing to CNS immune tolerance, can be detennined.
  • MBP27-42 recruits a reservoir of Foxp3+ rcgulatory T cells for the upkeep of CNS immune tolerance.
  • MBP27-42 specific T cells may enforce immune regulation through other regulatory mechanisms.
  • prior studies have demonstrated a role for Foxp3- T regulatory type 1 (TRI) cells in regulating autoimmunity, including EAE, through production of regulatory cytokines, including IL-10 and TGFP103.104.
  • TRI Foxp3- T regulatory type 1
  • intracellular cytokine staining for IL- 10 and TGFp is included when phenotyping MBP2742 specific T cells by flow cytometry.
  • IL- 1 Ofl/fl and CD4-CreERT2 are both commercially available and permit conditional deletion of IL-10 production in CD4+ T cells.
  • Aim 2 Neuropathology limits the presentation of homeostatic epitopes on MHC II molecules.
  • Hie lysate pre-cleared of debris by centrifugation will then be incubated with sepharose conjugated to an antibody specific to the MHC class II molecule.
  • peptides are eluted from MHC class II molecules with 10% acetic acid. Eluted peptides are then cleaned further with C 18 spin columns prior to being subjected to mass spectrometry and immunological analyses.
  • Aim 3 Therapeutic delivery of CNS derived endogenous peptides broadly safeguards against neuroinflammation-induced pathology
  • Exo-spinTM Using the Exo-spinTM system, EVs are precipitated and subjected through size exclusion chromatography to purify for EVs specifically. Confirmation is by immunoblot and ZetaView nanoparticle tracking analyzer prior to further downstream application. Following confinnation, exosomes undergo at least five freeze-thaw cycles to release intracellular contents; which is purified by C18 columns enriching for peptides. The collected material is subjected to mass spectrometry and immunological analyses.
  • Study #2 Assessing the therapeutic capacity of EVs containing MBP27-42 peptides to treat neuroinflammation
  • EVs arc enriched from DC2.4 cells, an immortalized dendritic cell line, with 100,000xg ultracentrifugation and confinn purity by immunoblot, TEM, and ZetaView nanoparticle tracking analyzer. Enriched vesicles are sonicated equally in the absence or presence of endogenous MBP27 42 and encephalitogenic MOG35-55 peptides.
  • Vesicles are further purified by both size exclusion chromatography and high-speed ultracentrifugation to remove any unincorporated peptides, effectively isolating intact control vesicles or vesicles containing our peptides of interest. Quality of enriched EVs is assessed similarly as mentioned above and further quantified by ZetaView nanoparticle tracking analyzer to control for the total number of vesicles injected into the CSF. Moreover, loaded peptides are fluorochrome conjugated enabling control for loading efficiency by nanoparticle flow cytometry. EAE is induced with MOG35-55 immunization in 8-week-old C57BL/6J mice.
  • control vesicles or vesicles containing either MBP27-42 or MOG35-55 are introduced into the CSF via intracistema magna (i.e.m.) injection on day 5 (prior to disease onset), day 10 (onset of paralytic symptoms), or day 16 (peak disease). All experimental groups with their respective littermate controls are subjected to standard EAE scoring for up to 25 days. In doing so, only the delivery of MBP27-42-containing vesicles across different time points is expected to exhibit therapeutic benefit, promoting the restoration of CNS immune tolerance. Furthermore, intravenous injection of EVs are also examined as it presents with a minimally invasive and convenient mode of delivery.
  • MBP27-42 an epitope abundantly presented on MHC II molecules, in regulating CNS immune homeostasis.
  • MBP2742 sufficiently suppressed against PLPw-isi-induced EAE in SJL/J mice.
  • the MBP27-42 sequence itself is highly conserved across different vertebrate species, including humans 106 107 . For these reasons, interrogation of the capability of MBP 2 7-42-containing vesicles to dampen neuroinflammation-induced damage in different active EAE models is sought.
  • mice receive i.c.m. injections of control vesicles or vesicles containing peptides on day 5 after which they are subjected to standard EAE scoring for up to 25 days.
  • MBP peptides are predicted to exhibit therapeutic efficacy at different stages of disease and in different strains of mice.
  • the observed suppression of EAE disease may remain restricted to i.c.m. delivery of MBP2742 at day 5, prior to the onset of paralysis.
  • EVs reflect the cell type and environment from which they are derived 108 109 , modulating the character of vesicles may bolster our antigen-specific immunotherapeutic approach.
  • a recent study demonstrated that vesicles derived from dendritic cell cultures treated with low dose IFNy reduced oxidative stress and enhanced CNS myelination processes in vivo 110 .
  • EVs derived from mesenchymal stem cells mildly but significantly reduced clinical EAE scores when administered intravenously at the peak of disease 11 this strengthens confidence in treating CNS autoimmunity according to the invention at a more advanced stage of disease.
  • MBP27-42 and control peptides are also loaded into vesicles derived from IFNy-stimulated dendritic cells and mesenchymal stem cells.
  • these studies further advance MBP27-42-containing vesicles as an antigen-specific immunotherapeutic for autoimmune disease.
  • CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS.
  • Interleukin-23 rather than interleukin- 12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744-748 (2003). https://doi.org: 10.1038/nature01355 Zhou, L. et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing ROR- gammat function. Nature 453, 236-240 (2008). https://doi.org: 10.1038/nature06878 Ichiyama, K. et al. Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through di-rect interaction with RORgammat. J Biol Chem 283, 17003-17008 (2008). https://doi.org: 10.
  • MHC I molecules place tyrosine residues at the end of the peptide binding pocket, often restricting the peptide length to 8-10 amino acid residues, although longer peptides bound to MHC I molecules have been reported.
  • the MHC II molecules are more open at the ends allowing for peptides to extrude beyond the confines of the binding cleft.
  • the MHC II molecules are more amenable and accommodate peptide lengths of around 15-25 amino acid residues.
  • the portions of the peptide not directly involved with the peptide binding groove of the MHC II molecule have been posited to play a role in influencing the stability of peptide binding. This adds an extra element of peptide association for MHC II molecules that of which is currently thought to be absent for MHC I molecules.
  • the cell pellet Prior to lysing, optionally wash the cell pellet (e.g., once with PBS buffer).
  • lysis buffer e.g., MEGA8/9 lysis buffer
  • protease inhibitors e.g., Roche protease inhibitor cocktail, PMSF, lodoacetamide, Leupeptin. Pipet up and down to thoroughly suspend cell pellet.
  • Lyse cells (e.g., for 1 hour in cold room while rocking).
  • wash samples e.g., as follows: a. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4; b. 5-10 mL 400 mM NaCl 20 mM Tris pH 7.4; c. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4: d. 5-10 mL 20 mM Tris pH 8.0)
  • Steps include:
  • Samples can analyzed using LC-MS/MS for identification of peptidome.
  • Example 3 Analyzing MHC I and II peptidomes in both humans and mice
  • peptidomes of antigens presented by MHC molecules in brain and lymphatic tissues of humans and mice were investigated by purification and identification using LC/MS. 14,609 peptides were identified in a protcomic assay of autoantigens presented on major histocompatibility complex classes I and II (MHC I and MHC II) in human and mouse and peripheral and CNS immune cells (i.e., SEQ ID NOS: 1-14,609). Peptides were analyzed for the following properties: a. whether shared by both humans and mice, or in humans only, or in mice only; b. binding to MHC I, to MHC II, or to both; c. enrichment in the CNS; and d. identification of the protein from which the peptide derives from, including beginning and end amino acids for the peptide (fragment) of the protein.
  • the central nervous system despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding conundrum.
  • CNS-derived endogenous regulatory (guardian) peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders.
  • guardian peptides During homeostasis, a preponderance of these guardian peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of guardian peptides was diminished. By then boosting the presence of these guardian peptides, a population of suppressor CD4+ T cells could be expanded and CNS autoimmune disease significantly reduced.
  • This unexpected discovery of CNS-derived autoimmune guardian peptides may be the molecular key adapting the CNS to receive information and to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.
  • the CNS seeks immunosurveillance yvhile actively providing immune cells with molecular cues to foster its tolerance to autoreactivity.
  • a thorough interrogation was performed of the CNS, its borders, and its draining lymph nodes for peptides bound to MHC II molecules.
  • CNS-de rived endogenous, regulatory self-peptides were identified, which were termed “guardian peptides”.
  • CD1 lb+ CD11c- macrophages were the predominant MHC Il-expressing cells (antigen-presenting cells, APCs) in the brain, which also includes the leptomeninges, and the dura
  • CD 19+ B cells were the main APCs in the deep cervical lymph nodes (dCLNs) and in tire superficial cervica lymph nodes (sCLNs) (FIGS. 16A-16E).
  • algorithmic predictions of MHC II- binding affinities utilizing three independent models 14 16 identified a propensity for peptides with relatively weaker affinities to be bound on MHC II molecules in the brain. Whereas, further downstream along the path of lymphatic drainage their binding affinities increased, suggesting heterogeneity to the repertoire of peptides bound to MHC II molecules in different compartments (FIG. 16F).
  • MBP peptides The abundant presentation of MBP peptides captivated our interest in understanding their potential function in the CNS during homeostasis. Identified MBP peptides were screened by immunizing with them to assess their encephalitogenicity. Whereas immunization of rodents with myelin oligodendrocyte glycoprotein (MOG)s5 55 induced paralysis ty pical of experimental autoimmune encephalomyelitis (EAE) 20 — a rodent model of multiple sclerosis (MS) 21 22 — immunization with endogenous MBP peptides, such as MBP160-175, did not induce paralysis (FIG. 14A).
  • MOG myelin oligodendrocyte glycoprotein
  • EAE experimental autoimmune encephalomyelitis
  • mice that had received MBPiso 175 peptides exhibited decreased cellularity relative to those immunized with MOG35 55 peptides (FIG. 14B).
  • MBP160-175 unlike MOG35-55, lacked an antigen-specific effector T cell response when assaying for IL-2 production by enzyme-linked immune absorbent sport (ELISpot) (FIG. 14C).
  • ELISpot enzyme-linked immune absorbent sport
  • mice were co-immunized with encephalitogenic MOG35-55 peptide and our endogenous MBP peptides.
  • the MBP160-175 sequence was directly modified by converting its arginine residues to citrulline.
  • T cells were individually sorted from draining lymph nodes of MOG35 55 only and of MOG35 55 + MBPiso 175 immunization groups, and then performed single-cell RNA sequencing (scRNA-seq) using the 10X Genomics Chromium -gene expression platform. Unsupervised clustering and dimensionality reduction was perfonned to project the cells into two dimensions using the uniform manifold approximation and projection (UMAP) algorithm.
  • UMAP uniform manifold approximation and projection
  • guardian MBP peptides may broadly apply to the treatment of neuroinflammatory as well as of neurodegenerative disorders.
  • MHC e.g.. MHC II
  • MHC II MHC Il-bound MBP peptides pervading the meningeal lymphatic network, extending from the brain parenchyma to the draining cervical ly ph nodes.
  • tissue-specific antigens are presented on MHC II molecules during homeostasis in their respective draining lymph nodes for the skin and intestines 36 as well as the pancreas 37 .
  • C57BL/6J (WT: JAX000664) and SJL/J (WT; JAX000686) were purchased from the Jackson Laboratory; mice were maintained in standard housing conditions (12 hour light/dark cycle and provided with sterilized water and regular rodent chow ad libitum unless stated otherwise). Mice were allowed to acclimate for at least one week in the animal facility prior to the beginning of any experiment. Adult males and females between 8-12 weeks of age were primarily used for our studies unless stated otherwise. Sample sizes were determined on the basis of a power analysis in accordance with previously published experiments. Experimenters, where necessary, were blinded to experimental groups during both scoring and quantification. All experiments were approved by the Institutional Animal Care and Use Committee at the Washington University in St. Louis. ii) Single Cell Isolations
  • mice were perfused with transcardial perfusion with PBS containing heparin (0.025%); samples were collected into ice-cold RPMI and maintained on ice for the entirety of tissue collection. Spleens were removed from the surrounding tissues and placed on ice-cold RPMI (Gibco) until further use.
  • lymph nodes i.e. deep cervical lymph nodes, superficial cervical lymph nodes, and inguinal lymph nodes
  • spleen isolations were performed following lethal i.p. injection of Euthasol. Subsequently, mice were perfused with transcardial perfusion with PBS containing heparin (0.025%); samples were collected into ice-cold RPMI and maintained on ice for the entirety of tissue collection. Spleens were removed from the surrounding tissues and placed on ice-cold RPMI (Gibco) until further use.
  • Tire whole spleen was digested and mashed through a 70 pm cell strainer with a glass pestle and washed with 5 mL of RPMI. Cells were then centrifuged at 450g for 5 minutes. RBC lysis was performed with 1 mL of ACK lysis buffer (Quality’ Biological); cells were incubated for 2 minutes and then 2 mL of ice-cold PBS was supplemented to the sample. Samples were subsequently centrifuged at 450g for 5 minutes and lysed red blood cells were aspirated. The cell pellets were then resuspended in FACS buffer (2% BSA.
  • EDTA ethylenediamine acetic acid
  • Lymph nodes were similarly digested and mashed through a 70 pm cell strainer, washed with 2 mL RPMI, centrifuged, resuspended in FACS buffer, and kept on ice until further use. Choroid plexi were removed from the brain; similarly, the dural meninges were peeled from the skull cap using Dumont #5 forceps (Fine Science Tools). Tire dural meninges were kept in ice-cold RPMI for the entirety of the tissue harvest.
  • Dural meninges were then digested for 15 minutes at 37°C with constant agitation using 1 mL of prewarmed digestion buffer (RPMI-1640 medium with 2% FBS, 1 mg/mL Collagenase VIII, and 0.5 mg/mL DNase I). They were then filtered similarly through a 70 pm cell strainer and provided with 1 mL of complete medium (RPMI with 10% FBS) to neutralize the enzymes. Samples were then centrifuged at 450g for 5 minutes, resuspended in FACS buffer and kept on ice. Lastly, brains and spinal cords were harvested and placed in ice-cold RPMI for the entirety of the collection.
  • prewarmed digestion buffer RPMI-1640 medium with 2% FBS, 1 mg/mL Collagenase VIII, and 0.5 mg/mL DNase I.
  • Brains were mechanically dissociated using sterile surgical scalpels into ⁇ 1 mm 3 cubes and digested with constant agitation in 2 mL of pre-warmed digestion buffer for 20 minutes at 37°C, triturated with a 10 mL serological pipette, digested for another 20 minutes, triturated with a 5 mL serological pipette, and digested for an additional 20 minutes.
  • a 1: 1 ratio of 22% bovine serum albumin (BSA) in PBS was added and centrifuged at 1000g for 10 minutes. Following centrifugation, the floating myelin layer was aspirated. Cell pellets were then neutralized with RPMI with 10% FBS to halt the enzymatic digestion.
  • BSA bovine serum albumin
  • Cells were isolated as described in '‘single cell isolations,” suspended in lysis buffer (40 mM MEGA 8, 40 mM MEGA 9, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM iodoacetamide, 20 pg ml— 1 leupeptin and Roche cOmplete Protease Inhibitor cocktail in phosphate-buffered saline), and rocked for 1 h at 4 °C. The cell lysate was spun in a centrifuge at 20,000g for 25 min at 4°C.
  • the supernatant was first incubated with polyclonal mouse immunoglobulin G (Bio X Cell; 1.5 mg antibody per sample) bound to Sepharose 4B at 4 °C for 30 min. The flow through containing peptide-MHC II complexes was collected, added to a tube containing phosphate-buffered saline-washed sepharose conjugated to the anti-I-A antibody (Y-3P;
  • Mass spectrometry scans were acquired in profile mode and tandem mass spectrometry scans were acquired in centroid mode, for ions with charge states 2-7, with a cycle time of 1.5 s.
  • Mass spcctromct ’ spectra were recorded from 375-1,500 Da at 120-K resolution (at m/z 200), and higher- energy collisional dissociation tandem mass spectrometry was triggered above a threshold of 2.0 x 104 . with quadrupole isolation (1.4 Da) at 15-K resolution and a collision energy of 30%. Dynamic exclusion was used (60 s), and monoisotopic precursor selection was on. vi) Mass spectrometry data analysis
  • EAE was induced in C57BL/6J mice by subcutaneous injection of MOG35-55 peptide (100 pg, CSBio) and/or different synthesized peptides including, MBP160-175, citrullinated MBPieo-ns. MBPiee-m. and MBPi 92-216 (GenScript) emulsified in Freund s adjuvant (Sigma .Aldnch) supplemented with 2 mg/mL of Mycobacterium tuberculosis (BD). Pertussis toxin (200 ng. List Biologicals) was injected i.p. on day 0 and day 2 following immunization with MOG and/or MBP.
  • EAE was induced in SJL/J mice by subcutaneous injection of PLP139-151 (100 pg, Vivitide) and/or different synthesized peptides including, MBP160 175, neurofilament light polypeptide (NEFL)i6o-i73 (GenScript) emulsified in Freund's adjuvant (Sigma Aldrich) supplemented with 2 mg/mL of Mycobacterium tuberculosis (BD). Pertussis toxin (400 ng, List Biologicals) was injected i.p. on day 0 and day 2 following immunization with PLP and/or MBP and NEFM.
  • PLP139-151 100 pg, Vivitide
  • NEFL neurofilament light polypeptide
  • GenScript neurofilament light polypeptide
  • BD Mycobacterium tuberculosis
  • 0 no clinical disease
  • 1 limp tail
  • 2 hindlimb weakness
  • 3 hindlimb paralysis
  • 4 partial front limb paralysis
  • 5 moribund.
  • mice were subcutaneously immunized with different peptides, including MOG35 55, MBP160-175, MBP166-185, and MBP192-216, among others, emulsified in Freund’s adjuvant.
  • 96- well polyvinylidene fluoride membrane plates (Millipore) were coated with IL-2 capture antibodies (BD Biosciences).
  • IL-2 capture antibodies (BD Biosciences). 7 days after immunization, cells were isolated from the draining lymph nodes and counted such that 5 x 105 cells would be loaded per well (each condition performed in triplicates). Cells were stimulated with their appropriate antigens and controls and incubated at 37°C in 5% CO2 for 24 hours.
  • IL-2 secreting cells were identified with IL-2 detection antibodies (BD Biosciences) and plates were developed following manufacturer’s directions (BD Biosciences). Spots quantified with CTL ImmunoSpot S6 Universal machines and Professional 6.0.0 software. ix) Single-cell RNA Sequencing.
  • the filtered and normalized matrix was used as input to the Seurat pipeline and cells were scaled across each gene, regressing out sequencing depth per cell, number of unique features, and percent mitochondrial reads, before the selection of the top 2,000 most highly variable genes using variance stabilizing transformation.
  • Principal Components Analysis was conducted and an elbow plot was used to select components for UMAP analysis and clustering.
  • Shared Nearest Neighbor (SNN) clustering optimized with the Louvain algorithm, as implemented by the Seurat FindClusters function was performed before manual annotation of clusters based on expression of canonical gene markers.
  • CD4 and CD8 cells were subset based on sub-cluster identity and gene expression and each subset was filtered to include genes that had at least 4 transcripts in at least 4 cells. Then the top 2000 highly variable genes were determined and included for further analysis using the SingleCellExperiment modelGeneVar and getTopHVGs functions. After filtering, limma and edgeR were used to build a model and conduct differential expression testing with the ImFit, contrasts.fit, and eBayes functions. Results were then filtered using a Benjamini- Hochberg adjusted p-value threshold of less than 0.05 as statistically significant.
  • CSV files containing single-cell transcript counts were downloaded from the Gene Expression Omnibus accession GSE118948, read into R and converted to matrices. Blood and choroid plexus tissue samples were discarded and the remaining leptomeningeal and parenchymal perivascular space samples were manually filtered for quality control. Filtering was performed to remove cells with less than 200 unique genes, less than 1,000 or greater than 25,000 transcripts, and mitochondrial transcript percentage greater than 30 percent. Expression values were then normalized using the scran and scater packages. The resulting log2 values were transformed to the natural log scale for compatibility with the Seurat (v3) pipeline42-44. Initial contamination in the fonn of lowly sequenced clusters were manually removed and the finalized subset of cells was scaled, underwent PCA, clustering, and annotation as described above.
  • the mean (or median) value of percentiles across all models was then used as the main source for the given antigen. We further represented these values as inverted percentile ranks such that larger values indicate peptides with increased likelihood to be a binder.
  • the decoy set was generated from the mouse proteome and consists of about 1 x 10 6 peptide fragments.
  • the DC2.4 cell line was purchased from the American Type Culture Collection (ATCC). Cells were maintained by culturing in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin/streptomycin (Gibco) and 2 mM L-glutamine (Gibco). All cells were grown in an incubator set at 37°C in 5% CO2. xiii) Extracellular vesicles isolation and preparation
  • the DC2.4 cells were cultured as stated above in complete DMEM medium in T-75 flasks to 80- 90% confluency. Cells were thoroughly washed with dPBS prior to replacing the medium with serum depleted DMEM. They were further cultured overnight. Supernatants were collected next day and spun at varying speeds, including 1.000g for 10 mins to rid of any cell contaminants as well as 20,000g for 30 mins to rid of any smaller debris. The supernatant, at this point, was further filtered with a 0.22 pm filter and ultracentrifuged at 100,000g for 2 hours to pellet down extracellular vesicles (EVs). All spins were performed at 4°C.
  • the pellet was resuspended in 500 pl PBS and visualized by either transmission electron microscope (JEOL JEM-1400, Phillips) or nanoparticle tracking analysis (ZetaView) to confirm their isolation, to measure their size, and to approximate the number of particles per milliliter
  • mice were anesthetized using ketamine/xylazine (100 mg/kg ketamine and 10 mg/kg xylazine). Their necks were shaved and cleaned appropriately with 70% iodine prior to placing their heads securely into the stereotactic frame. Ophthalmic solution was applied to prevent drying of their eyes prior to the procedure. Following this, a longitudinal incision of the skin was made at the back of their necks: the underlying muscles were retracted with hooks to expose the cistema magna. A 5pL Hamilton syringe with a 33-gauge needle was utilized to inject 5pL of PBS or extracellular vesicles (i.e.
  • Tire MHC-II peptidome of pancreatic islets identifies key features of autoimmune peptides. Nat Immunol 21, 455-463 (2020). Cohen, I. R. The cognitive paradigm and the immunological homunculus. Immunol Today 13, 490-494 (1992). Wucherpfennig, K. W. & Strominger, J. L. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80, 695-705 (1995). Lunemann, J. D. et al. EBNA1 -specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-gamma and IL-2.
  • Example 5 Assessment of Tubb 3, Map2, and Nefm as MHC guardian peptides in an EAE animal

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Abstract

Compositions and methods for the treatment, prevention, or reversal of a neuroinflammation-related disorder or autoimmune neural disorder in a subject in need in a patient in need are disclosed. The method includes administering a therapeutically effective amount of a composition that includes myelin basic protein (MBP) peptides.

Description

COMPOSITIONS AND METHODS OF TREATMENT FOR NEUROINFLAMMATION- RELATED DISORDERS
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[001] This invention was made with government support under a grant awarded by the National Institutes of Health (DPI AT010416). The Government has certain rights in the invention.
FIELD OF THE TECHNOLOGY
[002] The present disclosure generally relates to compositions and methods for the treatment, prevention, or reversal of neuroinflammation-related disorders using fragments of MHC -bound proteins.
BACKGROUND
[003] With incidence and prevalence on the rise, autoimmune diseases continue to debilitate countless individuals, increasing the healthcare burden while decreasing the quality of life. Current interventions, thus far, have focused on non-specific immunosuppressive therapies that come with substantial side effects. To that end, antigen-specific immunotherapies offer an appealing alternative to precisely target organ-specific autoimmunity. To personalize medicine, a deeper understanding of the repertoire of autoantigens presented on major histocompatibility complex (MHC) (e.g.. MHC class II) will direct the development of antigen-specific immunotherapies.
SUMMARY
[004] Provided herein is a pharmaceutical composition, which may comprise one or more peptides and a pharmaceutically acceptable excipient. Each of the one or more peptides may bind to a Major Histocompatibility Complex (MHC) molecule and may be a fragment of a protein selected from TABLE Cl, or a modified fragment thereof. Tire protein or peptide may be expressed at elevated levels in the central nervous system (CNS) relative to a control tissue. In one example, an expression level of the protein or peptide is 5 -fold higher than the control tissue. Alternatively, the protein may not be expressed at elevated levels in the CNS or may be expressed at decreased the levels in the CNS relative to a control tissue. In one example, the CNS tissue is dural meninges and the control tissue is a lymph node, such as a deep cervical lymph node or a superficial cervical lymph node. Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1- 14619, or a modified sequence thereof. [005] Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294. 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof. Each of the one or more peptides may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:597. 770, 1231, 1232, 1709, 1748. 2297. 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797. 4798, 4799, 4800, 4801, 4802, 4803, 4804. 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819. 4820. 4822, 4823, 4825, 4826, 4828, 4829. 5067. 5439, 5574, 5575, 14519, 14520. 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof. Each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, TABLE 8, TABLE 9, or TABLE 10, or a modified sequence thereof.
[006] Each of the one or more peptides may be a fragment of a myelin basic protein (MBP). tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM). Each fragment of MBP may independently comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754. 10755, 10756, 10757, 10758. 10759, 10760, 10761, 10762, 10763. 10764, 10765, 10766,
10767, 10768. 10769. 10770, 10771, 10772. 10773. 10774, 10775, 10776, 10777. 10778, 10779, 10780,
10781 , 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794,
10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808,
10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822,
10823, 10824, 10825, 10826, 10827, 10828. 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836,
10837, 10838. 10839, 10840, 10841, 10842. 10843, 10844, 10845, 12162, 14537. 14538, 14539, 14540,
14541, 14542. 14543. 14544, 14545, 14546. 14547. 14548, 14549, 14550, 14551. 14552, 14553, 14554,
14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568,
14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582,
14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596,
14610, 14611, 14612, and 14613. At least one of the one or more peptides may comprise or consist of the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
[007] The pharmaceutical composition may further comprise one or more extracellular vesicles (EV) or liposomes enclosing the one or more peptides. The one or more EV may be an exosome, a microvesicle, or an apoptotic body of a cell, optionally a human cell. The one or more EV may oligodendrocyte- derived. optionally from a dendritic cell, which may be of the cell line DC2.4. The one or more EV may be synthetic.
[008] Provided herein are one or more nucleic acids encoding the one or more peptides. One or more vectors may each comprise at least one of the one or more nucleic acids. The vectors may be viral vectors. Tire viral vectors may be derived from an adenovirus, adeno-associated virus, or retrovirus, optionally selected from a lentivirus. Tire vectors may be non- viral vectors.
[009] The one or more nucleic acids may be mRNAs. The mRNAs may be non-replicating mRNAs, self-amplifying mRNAs (saRNAs), or circular RNAs (circRNA). Tire mRNAs may be non-replicating mRNAs; and each rnRNA may comprise a coding region encoding one of the one or more peptides, a first UTR, a second UTR, a 5’ cap. and a poly(A) tail; and each coding region may be flanked by the UTRs. Each 5’ cap may comprise a 7-methylguanosine (m7G) cap that is connected to a first nucleotide of each mRNA (N) by triphosphates (ppp) to form m7GpppNp. Each m7G cap may comprise a methylated 2 ’-OH on the first nucleotide connecting the 5’ end of each mRNA to the cap (capl or m7GpppNimp). Each m7G cap may comprise a methylated 2 ’-OH on the first nucleotide and a second nucleotide connecting the 5’ end of each mRNA to the cap (cap2 or m7GpppNimpN2mp). Each mRNA may comprise one more modified nucleosides, optionally selected from the group consisting of pseudouridine (t|/), N1- methylpseudouridine (mfy). 5 -methoxyuridine (mo5U), 2-thiouridine (s2U). 5 -methylcytidine (m5C) and N6-methyladenosine (m6A). The nucleic acids may be encapsulated, optionally by lipid nanoparticles (LNPs), polyplexes, polymeric nanoparticles, lipopolyplexes (LPPs), or cationic polypeptides. The pharmaceutical composition may comprise the one or more nucleic acids, one or more vectors, or one or more mRNAs.
[010] Provided herein are the one or more peptides or the pharmaceutical composition for use in treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury'. Also provided herein are a method of treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury in a subject in need thereof, which may comprise administering the one or more peptides or the pharmaceutical composition to the subject: and use of the one or more peptides or the pharmaceutical composition in the manufacture of a medicament for ting, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury. Hie peptides or pharmaceutical composition may be or may be intended to be administered to a subject by infusing or injecting into the cerebrospinal fluid (CSF) of the subject.
[Oi l] Hie neuroinflammation-related disorder or an autoimmune neural disorder may be selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN). acute optic neuritis (AON). Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cold agglutinins, and disialosyl antibodies (CANOMAD), chronic meningitis, Behcet's disease , Central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillar acidic protein (GFAP), Hashimoto’s encephalitis, hypertrophic pachymeningitis, lgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurologic syndrome (PNS), Parkinson’s disease (PD), steroid responsive encephalopathy associated with autoimmune thyroiditis (SREAT). stiff person syndrome, Susac’s syndrome, and transverse myelitis. The acute CNS injury may be selected from the group consisting of a spinal cory injury, traumatic brain injury, spinal cord injury', optic nerve injury', and stroke.
BRIEF DESCRIPTION OF THE DRAWINGS
[012] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. Hie drawings are not intended to limit the scope of the present teachings in any way.
[013] FIGS. 1A-1B show that MHC II presentation in the CNS centers on a particular MBP region.
FIG. 1A demonstrates tire composition of MHC Il-bound peptides derived from proteins labeled as CNS- enriched. FIG. IB shows the exact sequences of peptides bound to MHC II molecules deriving from MBP. [014] FIGS. 2A-2D show that MBP peptides gamer regulatory T cells to suppress CNS autoimmunity. FIG. 2A depicts a schematic for the induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. FIG. 2B shows clinical EAE score tracked over 21 days for mice immunized with either MOG35-55, MOG35-55 and MBP33-52, or MOG35-55 and MBP (27-42 subscript); two-way ANOVA with Sidak’s multiple comparison test, p-values, where significant, indicated on plots. FIG. 2C displays representative flow cytometry plots with associated quantifications as in FIG. 2D for evaluation of regulatory T cells in the deep cervical lymph nodes (dCLNs) when comparing between MOG3555 as well as MOG35-55 and MBP2742 co-immunized groups; unpaired two-tailed Student’s t-test, p-values indicated where relevant.
[015] FIGS. 3A-3B show that presentation of MBP peptides is generalizable and similarly suppresses neuroinflammation in a different active EAE model. FIG. 3A depicts CNS-enriched peptides identified across both the brain and dural meninges in SJL/J male mice as well as in fresh frozen human dura. FIG. 3B shows clinical EAE scores tracked over 21 days for SJL/J mice immunized with PLP139-151, PLP139-151 and NEFL160-173, or PLP139-151 and MBP27-42. Statistics done with two-way ANOVA with Sidak’s multiple comparison test, p-values, where significant, indicated on plots..
[016] FIG. 4 show s the harnessing of MBP peptides as a therapeutic against CNS autoimmunity. Schematic displaying the purification of extracellular vesicles (EVs) and their subsequent packaging with peptides via sonication. Fluorescently-labeled (Alexa Fluor 488) ovalbumin (OVA) incorporation into vesicles and cellular uptake confirmed by flow cytometry. Visualization of EVs by both electron microscopy (EM) and ZetaView7 nanoparticle tracker visualization. Clinical EAE score tracked over 21 days for mice induced for EAE and with introduction of either empty EVs or MBP27-42 containing EVs via intracistcma magna (i.e.m.) injection prior to onset of disease; two-w ay ANOVA and p-valuc indicated on plot. Peak EAE scores for both conditions also quantified and displayed on the right, unpaired two- tailed Student’s t-test, p-value indicated on plot.
[017] FIGS. 5A-5B show7 analysis of the MHC II peptidome. FIG. 5A depictsepitope mapping by Gibbs cluster analysis of MHC-II-bound peptides of 8-week-old C57BL/6J.I-Ab mice. FIG. 5B depicts distribution for MHC-II-bound peptides enriched for proteins that are expressed at elevated levels in tire CNS. ‘"Other” represents the sum of CNS elevated proteins with only a single MHC-II-bound peptide identified. These include: MAP2, NEFL, DPYSL2, CNP, ISLR2, BCAS1, IRS4, PTGDS, and TBC1D30. [018] FIG. 6A depicts the experimental scheme for induction of experimental autoimmune encephalomyelitis (EAE). FIG. 6B depicts average clinical EAE score in C57BL/6J mice immunized with MOG33-55 (red), MBP12-26 (gray), or MBP2742 (blue) on day 0; mice were intraperitoneally injected with pertussis toxin (PTx) on day 0 and day 2. Standard EAE scoring to assess mice daily for 20 days post immunization. N = 5 mice per group, p-value is indicated on the graph.
[019] FIGS. 7A shows average clinical EAE score in C57BL6/J mice immunized with MOG35-55 (red), MOG35-55 + MBP12-26 (gray). MOG35-55 + MBP27-42 (blue), or MOG35-55 + MBP2742 (citrullinated) (yellow) on day 0; standard EAE scoring to assess mice daily for 20 days post immunization. FIG. 7B shows peak EAE score for C57BL6/J mice as immunized in FIG. 7A. N = 5 mice per group, p-values are indicated on the graphs.
[020] FIGS. 8A-8D show mice immunized with MOG35-55 or MOG35-55 + MBP27-42 to induce EAE (N = 4 per group). dCLNs and brain meninges assessed by flow cytometry at day 13 and 16 respectively post induction of EAE. Representative flow plots gated on live, CD45+ , TCR[3+ , CD4+ , showing Foxp3 and RORyt with frequencies indicated for the dCLNs (FIG. 8A) and brain meninges (FIG. 8C). Summary data showing RORyt and Foxp3 expression as a frequency of CD4+ T cells for the dCLNs (FIG. 8B) and brain meninges (FIG. 8D), p-valucs indicated on individual plots.
[021] FIG. 9A shows average clinical EAE score in SJL/J mice immunized with PLP139-151 (red), PLP139 151 + NF-L160 173 (gray), or PLP139 151 + MBP 2742 (blue) on day 0* standard EAE scoring to assess mice daily for 21 days post immunization. FIG. 9B shows peak EAE score for SJL/J mice as immunized in FIG. 9A. N = 5 mice per group, p-values are indicated on the graphs.
[022] FIG. 10A shows an immunoblot performed on both supernatant (SN) and pellet post high-speed ultracentrifugation evidencing enrichment of tetraspanins CD9 and CD63, markers of extracellular vesicles, in the pellet. FIG. 10B shows a negative stain by transmission electron microscopy of enriched extracellular vesicles. FIG. 10C shows an experimental scheme (top) depicting tire insertion of MBP27-42 peptides into extracellular vesicles prior to intracistema magna (i.e.m.) injection. Average clinical EAE score in C57BL/6J mice immunized with MOG35-55 that received either empty vesicles (red) or MBP2742- containing vesicles (blue) via i.e.m. injection on day 5; standard EAE scoring to assess mice daily for 21 days post immunization. N = 5 mice per group, p-value is indicated on the graphs.
[023] FIG. 11A shows average clinical EAE score in C57BL/6J mice immunized with MOG35-55 (red) or CFA only (black) on day 0; standard EAE scoring to assess mice daily for 16 days post immunization. FIG. 11B depicts representative images and FIG. 11C depicts quantification of percent coverage of intracistema magna (i.e.m.) injected OVA647 protein in the dCLN of CFA only or CFA + MOG35-55 immunized mice at peak of EAE disease (day 16). Scale bar, 150 pm. P-values indicated on plots.
[024] FIGS. 12A-12C depicts binding motifs for both MHC I alleles H2-Kb (FIG. 12A)and H2-Db (FIG. 12B)as well as the sole MHC II allele H2-Ab (FIG. 12C) in C57BL/6J mice as predicted by both the netMHCpan and netMHCIIpan algorithms.
[025] FIGS. 13A-13E show that the CNS MHC II peptidome reveals presentation of endogenous CNS peptides. FIG. 13A depicts a schematic illustrating the mass spectrometric identification of MHC II- bound peptides from brain (including leptomeninges), dural meninges (dura), and lymph nodes, including the deep cervical lymph nodes (dCLNs) and superficial cervical lymph nodes (sCLNs), of C57BL/6J male mice. FIG. 13B shows assessment of the proportion of total unique identified peptides that could be designated as CNS enriched (teal bar); percentages indicated above each individual bar. FIG. 13C depicts a donut plot representation of CNS-enriched, MHC Il-bound peptides identified for each individual tissue. The percent of which is composed by MBP is indicated where relevant. FIG. 13D shows a Venn diagram depicting the relationship between CNS-enriched, MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs. FIG. 13E shows a summary of individual peptide sequences contained within the MBP158-195 region as defined by the MHC II peptidome.
[026] FIGS. 14A-14K show that MBP is non-encephalitogenic and fosters immunosuppression. FIG. 14A show s average clinical EAE scores assessed by immunization with either MOG3555 or MBPieo 175 in C57BL/6J mice (n=5 mice per group). Plots display mean ± s.e.m and represent two independent experiments. P-value indicated on plot and determined by two-way ANOVA. Mice immunized with either MOG35 55 or MBPieo-ns and draining (inguinal) lymph nodes were extracted at day 7 post immunization (n=3 mice per group). FIG. 14B shows quantification of cell counts (mean ± s.e.m, unpaired two-tailed Student’s t-test). FIG. 14C shows an ELISpot assay performed to measure IL-2 production by CD4+ T cells upon recall with peptides as indicated on the x-axis. Data shown as mean ± s.e.m, two-way ANOVA with Sidak’s multiple comparisons test. FIG. 14D shows average clinical EAE scores assessed by immunizing with MOG35-55 alone or co-immunizing either MOG35-55 and citrullinated MBP160 175 (MBP160 17 (cit.)) or MOG35 55 and MBP160-175 in C57BL/6J male mice (n=5 mice per group). Plots display mean ± s.e.m and represent three independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test. FIG. 14E depicts a UMAP visualization of T cells from the draining (inguinal) lymph nodes of MOG35-55 immunized or MOG35-55 and MBP160-175 co-immunized C57BL/6J mice. FIG. 14F shows Log2 fold change determined across the different identified clusters by scRNA-seq when comparing MOG35 55 to MOG35 -55 and MBP160 175 immunizations. Significant differences highlighted on the plot. FIG. 14G shows gene ontologies identified for CD4+ (left) or CD8+ (right) T cells that were found to be significantly upregulated in the MOG35-55 and MBPiso 175 over the MOG35-55 group. FIGS. 14H-14I depict representative flow cytometry plots: with associated quantifications (FIG. 14H) and depicting the proportion of CTLA-4+ Foxp3- unconventional suppressor T cells or Foxp3+ regulatory T cells (Treg) within the CD4+ T cell population in the dCLN 13 days post-immunization (FIG. 141) (n=3 mice per group, mean ± s.e.m, impaired two-tailed Student’s t-test). FIGS. 14J-14K show epresentative flow cytometry plots (FIG. 14J) with relevant quantification (FIG.14K) demonstrating the proportion of CTLA-4+ Foxp3- CD39+ within the CD4+ T cell population in the spinal cord 13 days postimmunization (n=5 mice per group, mean ± s.e.m. unpaired two-tailed Student’s t-test).
[027] FIGS. 15A-15F show that therapeutic delivery of guardian MBP peptides guards against CNS autoimmunity. FIG. 15A depicts a schematic depiction of isolating peptides bound to MHC II molecules in the brain, dura, and spinal cord at the peak (day 16) of MOG35-55 induced EAE. FIG. 15B shows the MHC II peptidomes from EAE and naive mice. Quantified relative abundances for peptides was identified within different antigenic regions to generate a heatmap, illustrating alterations to the repertoire of MHC Il-bound peptides in EAE when compared to naive mice. Relative abundances (normalized peak areas) of indicated peptides in heatmap were measured relative to (Cblni57-72) for the brain and (Mbpiss- 236) for the dura. FIG. 15C depicts a bar graph representation of the relative abundances of MBP peptides contained within the MBP158-195 or MBP196-236 regions for the brain (top) and dura (bottom) when comparing EAE to naive mice. Relative abundances (normalized peak areas) were measured relative to (Cbln 15772) for the brain and (Mbpi96-23e) for the dura. FIG. 15D shows an experimental design depicting i.e.m. injection of extracellular vesicles (empty, MBP160-175, or MBPi6o-i75(cit.)) after which duras were assessed by flow cytometry two days later, e, Representative flow cytometry plots (top) with associated quantifications (bottom) revealing the proportion of cells expressing CTLA-4+ Foxp3-unconventional suppressor T cells (left) or Foxp3+ Treg (right) within the CD4+ T cell population (n=5 mice per group, mean ± s.e.m. one-way ANOVA with Tukey’s multiple comparison test). FIG. 15F depicts an experimental scheme atop illustrating i.e.m. injection of PBS, MBP160-175 extracellular vesicles, or MBPi6o-i75(cit.) extracellular vesicles 7 days after immunization with MOG35-55 to induce EAE in C57BL/6J mice. Average clinical EAE scores assessed until day 20 post immunization (n=5 mice per group). Plots display mean ± s.e.m and represent three independent experiments. P-value indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test. [028] FIGS. 16A-16H show characterization of the MHC II peptidome in the CNS of C57BL/6J male mice. FIG. 16A shows peptide length distribution represented as a percent of the total number of unique peptides identified by the MHC II peptidome for the brain (which also includes the leptomeninges), dura, dCLNs, and sCLNs. FIGS. 16B-16D show representative flow cytometry plots depicting the gating strategy used to identify the distribution of MHC II-expressing cells. Within the MHC 11+ gate. B cells (gated on CD19+ CD11c- ), dendritic cells (DCs, gated on CD19- CD1 lc+ ), and macrophages (MO, gated on CD19- CD11c- ) were identified. Macrophages were further confirmed by F4/80+ and CD1 lb+ staining (not shown). To the right, bar plots show the frequency of the aforementioned antigen presenting cells as a percent of CD45+ MHC 11+ cells for the brain (FIG. 16B), dura (FIG. 16C), dCLNs (FIG. 16D), and sCLNs (FIG. 16E). FIG. 16F shows a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides. The median is represented by a solid line and the first as well as the third quartiles are represented by dashed lines. P-values indicated on plot (one-way ANOVA with Tukey’s multiple comparison test). FIG. 16D shows a Venn diagram depicting the relationship between all unique MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs. FIG. 16H shows a summary of individual peptides identified on the MBP sequence as defined by the MHC II peptidome of C57BL/6J male mice.
[029] FIGS. 17A-17F show the characterization of the MHC II peptidome in the CNS of C57BL/6J female mice. FIG. 17A shows a pie chart defining the makeup of CNS-enriched, MHC Il-bound peptides across each individual tissue. Percent of CNS-enriched peptides that are MBP are indicated in the plots, where relevant. FIG. 17B shows a summary of all identified individual peptide sequences derived from MBP in the C57BL/6J female MHC 11 peptidome. FIG. 17C depicts a Venn diagram representation of individual overlapping CNS-enriched peptide sequences identified between C57BL/6J females and males in the brain (including leptomeninges). FIG. 17D depict a bar graph showing the relative abundance of MBP peptides in the brain that comprised either the MBPiss-iss or MBP196-236 regions in both C57BL/6J males and females. Relative abundances (normalized peak areas) measured against a common identified peptide sequence, Dagl488-531, from which relativity was ascertained. FIG. 17E depicts a Venn diagram representation of individual CNS-enriched overlapping peptide sequences identified between C57BL/6J females and males in the dura. FIG. 17F depicts a bar graph showing the relative abundance of MBP peptides in the dura that comprised either the MBP158-195 or MBP196-236 regions in both C57BL/6J males and females. Relative abundances (normalized peak areas) measured against a common identified peptide sequence, Sptnfsi-ssg, from which relativity was detennined. [030] FIGS. 18A-18F show the characterization of the MHC II peptidome in the CNS of of SJL/J male mice. FIG. 18A depicts a violin plot depicting predicted binding affinities for unique MHC Il-bound peptides in the brain (which also includes the leptomeninges), dura, dCLN, and sCLN of SJL/J mice. The median is represented by a solid line, and the first as well as the third quartiles are represented by dashed lines. P-values indicated on plot (one-way ANOVA with Tukey’s multiple comparison test). FIG. 18B shows evaluation of the proportion of total unique identified peptides designated as CNS enriched (teal bar) in the brain, dura, dCLN, and sCLN; percentages indicated above each individual bar. FIGS. 18C- 18D depict a Venn diagram illustrating the relationship between all peptides (FIG. 18C) or CNS-enriched peptides (FIG. 18D) bound to MHC II molecules across the different tissues in SJL/J male mice. FIG. 18E depicts a donut plot representation of all CNS-enriched peptides identified in the MHC II peptidome of SJL/J male mice for the brain, dura, and sCLN. The part of the whole for which MBP represents is indicated in the plot, where relevant. FIG. 18F shows a depiction of all individual peptide sequences deriving from MBP identified in the SJL/J male MHC II peptidome.
[031] FIGS. 19A-19E show that endogenous guardian MBP peptides protect across different models of neuroinflammation. FIG. 19A depicts an experimental design of immunizing either C57BL/6J or SJL/J mice with MOG35-55 or PLP139-151 peptides, respectively, with or without MBP or NEFL peptides to actively induce EAE. Separately, C57BL/6J mice were also immunized individually with myelin associated peptides to perform ELISpot assay. FIG. 19B shows that mice immunized with either MBPI66. 185 or MBP192-216 and draining (inguinal) lymph nodes were extracted at day 7 post immunization (n=3 mice per group). ELISpot assay performed to measure IL-2 production by CD4+ T cells upon recall with peptides as indicated on the x-axis. Data shown as mean ± s.e.m, two-way ANOVA with Sidak’s multiple comparisons test. FIG. 19C shows average clinical EAE scores assessed by immunizing with MOG35-55 alone or co -immunizing either MOG35-55 and MBPies-iss or MOG35-55 and MBP192-216 in C57BL/6J male mice (n=5 mice per group). Plots display mean ± s.e.m and represent two independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test. FIG. 19D shows average clinical EAE scores assessed by immunizing with MOG35-55 alone or co-immunizing either MOG35-55 and MBPieo-ns (cit.) or MOG35 55 and MBPieo-ns in C57BL/6J female mice (n=5 mice per group). Plots display mean ± s.e.m and represent three independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test. FIG. 19E shows average clinical EAE scores assessed by immunizing with PLP139 151 alone or co-immunizing either PLP139 151 and MBP160-175 or PLP139 151 and NEFL160-173 in SJL/J male mice (n=5 mice per group). Plots display mean ± s.e.m and represent two independent experiments. P-values, where significant, indicated on plot and determined by two-way ANOVA with Sidak’s multiple comparisons test.
[032] FIGS. 20A-20E show that peripheral presentation of MBP peptides induces conventional Tregs. FIG. 20A depicts a dot plot of population markers from single cell RNA-sequencing scaled by percentage of cells expressing marker genes for each T cell cluster. FIG. 20B shows a representative gating strategy for flow7 cytometry used to define within CD4+ T cells, CTLA-4+ Foxp3- suppressor T cells as well as conventional Foxp3+ regulatory T cells (Treg). CTLA-4+ Foxp3- CD4+ T cells further gated to assess CD39. PD-1 (CD279), and IL-10 expression. FIGS. 20C-20D show representative flow cytometry plots with relevant quantifications (FIG. 20C) demonstrating the proportion of CTLA-4+ Foxp3- suppressor T cells and Foxp3+ Treg within the CD4+ T cell population in the inguinal (draining) lymph nodes 13 days post-immunization (FIG. 20D) (n=3 mice per group, mean ± s.e.m, unpaired two-tailed Student’s t-test). FIG. 20E shows quantification of conventional Foxp3+ Treg as a frequency of CD4+ T cells in the spinal cord 13 days post-immunization (n=5 mice per group, mean ± s.e.m, unpaired two-tailed Student's t-test).
[033] FIGS. 21A-21G show7 that neuroinflammation alters the MHC Il-bound repertoire of autoantigens. FIGS. 21A-21B show that mice immunized with CFA alone or CFA + MOG35-55 to assess for changes to MHC II expression as shown by representative flow7 cytometry plots (FIG. 21A) and associated quantifications (FIG. 21B), n=5 mice per group, mean ± s.e.m, unpaired two-tailed Student’s t-test. FIG. 21C show s a pie chart depiction of CNS-enriched, MHC Il-bound peptides identified in the brain and spinal cord at the peak of EAE disease. Below is a visual summary of individual MBP sequences identified. FIG. 21D shows a population pyramid representation of the distribution of amino acids identified at the C -terminus as a percent of the total MHC Il-bound peptides, indicating cleavage preference between EAE and naive mice. FIG. 21E shows UMAP projections of the Jordao ct al. dataset displaying broad cell lineages, color-coded accordingly, when analyzing naive, pre-symptomatic, and EAE mice. eDCs = conventional dendritic cells. migDCs = migratory dendritic cells. FIG. 21F shows a volcano plot depicting differences in gene expression when comparing microglia in EAE-induced mice to controls. Arrows indicate peptidases that could be identified. FIG. 21G shows gene ontologies found to be significantly upregulated in microglia when comparing between EAE and naive mice.
[034] FIGS. 22A-22F show that delivery of encapsulated MBP peptides directly into the CSF induces unconventional suppressor CD4+ T cells. FIG. 22A shows an immunoblot performed on the supernatant (SN) and resuspended pellet after ultracentrifugation evidencing enrichment of tetraspanins, CD9 and CD63, markers of extracellular vesicles (EVs). Below7 is a negative stain by transmission electron microscopy depicting enriched EVs, scale bar indicated on the panel. FIG. 22B shows that gating strategy for flow cytometry analysis to define within CD4+ T cells, CTLA-4+ Foxp3- unconventional suppressor T cells as well as conventional Foxp3+ Treg. At tire bottom, representative flow plots of the dCLNs depicting gates for these populations with i.c.m. injection of empty EVs, MBP160-175 EVs, MBP160-175(cit.) EVs. FIGS. 22C-22E show quantification of unconventional CTLA-4+ Foxp3- suppressor T cells as well as conventional Foxp3+ Treg as a frequency of CD4+ T cells of the deep cervical lymph nodes (FIG. 22C), superficial cervical lymph nodes (FIG. 22D), and spleen (FIG. 22E). FIG. 22F shows mice were immunized with MOG35 55 to induce EAE after which they were provided with PBS or free MBP160-175 peptides via i.c.m. injection on day 7. Average clinical EAE scores assessed until day 20 post immunization (n=5 mice per group). Plots display mean ± s.e.m and represent two independent experiments. P-value indicated on plot and detennined by two-way ANOVA.
[035] FIGS. 23A-23G show candidates for regulatory peptides against CNS autoimmunity. Clinical EAE scores tracked over 19 days post immunization with MOG35-55 or MOG35-55 and Tubb3 peptide 1 (FIG. 23A); MOG35-55 or MOG35-55 and Tubb3 peptide 2 (FIG. 23B); MOG35-55 or MOG35-55 and Tubb3 peptide 3 (FIG. 23C); MOG35.55 or MOG35-55 and Tubb3 peptide 4 (FIG. 23D); MOG35-55 or MOG35-55 and Map2 (FIG. 23E); MOG35-55 or MOG35-55 and Neftn peptide 1 (FIG. 23F); MOG35-55 or MOG35-55 and and Nefm peptide 2 (FIG. 23G). Statistics done with two-way ANOVA, asterisks indicating p-values shown on plot (* if p<0.05, ** if p<0.01, *** if p<0.001, **** if p<0.0001),
DETAILED DESCRIPTION
[036] While autoreactive T cells are well-known for provoking autoimmune disease, their protective roles in limiting tissue damage have been described in heart, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer’s disease, stroke, and traumatic injury, among others. With evidence supporting a suppressive and beneficial function for autoreactive T cells, the molecular and cellular mechanisms interconnecting the CNS and the peripheral immune system requires further investigation.
[037] This search has benefited from the recent rediscovery of a bona fide lymphatic network in the meninges, the membranous coverings that envelop the CNS. Emphasizing a role for the brain borders as an intimate space for interactions between CNS-resident and peripheral immune cells, the brain meninges provide a landscape of opportunities to examine the local presentation of autoantigens to autoreactive T cells. [038] Moreover, meningeal lymphatic vessels serve as conduits for the drainage of antigens and cells to secondary' lymphoid organs, specifically the deep cervical lymph nodes (dCLNs). For these reasons, the inventors had the insight to examine CNS-specific autoantigen presentation in CNS-associated tissues and how it contributes to immunosurveillance will inform novel mechanisms through which autoantigens maintain tissue homeostasis and protect against overt autoimmunity.
[039] Tire compositions and methods of the present disclosure are based on the surprising discovery that in an experimental MOG35-55 -induced autoimmune encephalomyelitis (EAE) model, co-immunization of MOG35-55 with MBP33-42 peptides ameliorated EAE disease. Further analyses by flow cytometry prior to the peak of EAE disease unveiled an elevated frequency of regulatory Foxp3-expressing CD4+ T cells in CNS-associated tissues of mice coimmunized with the MBP peptides. Moreover, therapeutic delivery of extracellular vesicles packaged with MBP peptides into tire cerebrospinal fluid (CSF) similarly suppressed MOGss-ss-induced EAE disease when compared to control vesicles without MBP peptides.
[040] Throughout the specification, fragments of MBP may be described at certain times according to traditional amino acid positional numbering, and at other times according to Goli number. As would be recognized by a skilled artisan, the MBP3342 fragment is equivalent to the MBP166-175 fragment, when accounting for Goli numbering differences.
[041] The data forming the basis for this disclosure took advantage of a specialized platform for immunopeptidomics, spearheading our understanding of the CNS MHC peptidome (e g., MHC I and MHC II). In doing so, we identified an abundance of peptides derived from myelin basic protein (MBP) that centered around a common sequence, MBP3342. Without being limited to any particular theory, it is thought that the presentation of endogenous CNS autoantigens gives rise to a reserve of regulatory T cells critical for the maintenance and restoration of CN S immune tolerance in both physiology and pathology, respectively.
[042] Tire CNS antigen repertoire used to develop the compositions and methods of the present disclosure overcome the shortcomings of existing methods that have hampered our capacities to efficaciously employ antigen-specific immune -tolerizing therapeutics. Inadequate knowledge of the repertoire of antigens presented during homeostasis of the CNS is one barrier overcome by utilizing an optimized technique to unravel native peptides bound to major histocompatibility complex class II (MHC II) molecules in the CNS and its borders. In addition, rather than focusing on a limited number of peptides to investigate autoreactive T cells, as has been done in previous methods, the present disclosure extensively characterizes the MHC II peptidome in CNS during homeostasis, providing the ability to broadly understand the specificities of autoreactive T cells in the CNS. Moreover, the identified peptides described herein are useful in directing the development of MHC II tetramers, allowing for tracking and the development of phenotype autoreactive CNS-specific T cells. The methods described herein provide insights into the function of relevant antigen presentation in the CNS to harbor local populations of autoreactive T cells - an understanding of which illuminates mechanisms the CNS employs to acquire immune privilege.
[043] Another critical barrier to the development of antigen-specific immunotherapies derives from an insufficient understanding of disease-initiating autoantigens and subsequent epitope spreading often arising as a consequence of chronic autoimmune or inflammatory response. The present disclosure overcomes this barrier by leveraging an established platform to define the MHC II peptidome to understand how the repertoire of autoantigens presented on MHC molecules diverges from homeostasis with neuroinflammation. Further, existing antigen-specific therapeutic strategies also face a barrier of being constrained to identified pathogenic epitopes and finding ways to manipulate either their sequence or their delivery to drive tolerogenic immune responses. The methods disclosed herein circumvent this barrier by taking advantage of the knowledge of CNS-derived endogenous antigen presentation in homeostasis to inform antigen-specific immunotherapies. Thus, in contrast to targeting pathogenic autoantigens to directly tolerize against, the disclosed methods therapeutically deliver a homeostatic, CNS-specific, MHC Il-bound autoantigen to suppress and protect against CNS autoimmunity.
[044] The compositions and methods described herein provide an expanded comprehension of the repertoire of autoantigens presented and of autoreactive T cells within the CNS, thus furthering understanding of the mechanisms supporting CNS immune privilege and immune tolerance by extension. Tire methods disclosed herein foster an understanding that will guide antigen-specific immunotherapies, enabling the induction of immune tolerance in other tissues to protect against allo- or autoimmune attack for clinical benefit.
1. Definitions
[045] So that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.
[046] The terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. Thus, the term “and/or” as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).
[047] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises.” “comprising,” “has,” “having.” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
[048] The term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ± 5%, but can also be ± 4%, 3%. 2%,1%, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
[049] As used herein, the term “subject” refers to a mammal, preferably a human. The mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.
[050] As used herein, the tenn “control population,” “normal population” or a sample from a “healthy” subject refers to a subject, or group of subjects, who are clinically determined to not have a disease. [051] The terms “treat,” “treating,” or “treatment” as used herein, refers to the provision of medical care by a trained and licensed professional to a subject in need thereof. The medical care may be a diagnostic test, a therapeutic treatment, and/or a prophylactic or preventative measure. The object of therapeutic and prophylactic treatments is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results of therapeutic or prophylactic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder orthose in which the disease, condition or disorder is to be prevented. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of disease.
[052] Discussed below are components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and pennutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules of the compound are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E. B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. [053] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of tire group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[054] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
[055] No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
2. Molecular Engineering
[056] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
[057] The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, tire use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
[058] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
[059] A '‘promoter” is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near tire start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
[060] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit the translation of a specific RNA molecule of interest. For the practice of tire present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
[061] The '‘transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein-encoding sequences in tire 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
[062] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
[063] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
[064] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e g., the 3 '-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
[065] Tire term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in a genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".
[066] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or plant into which a heterologous nucleic acid molecule has been introduced. Tire nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransfonned" refers to normal cells that have not been through the transformation process. [067] "Wild-type" refers to a virus or organism found in nature without any known mutation.
[068] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein are within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
[069] Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Mcgalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X/Y100, where X is tire number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B. the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
[070] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by He, Leu by He, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium- containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art- known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
[071] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed.. Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
[072] TABLE 1: Conservative Substitutions I
[073] TABLE 2: Conservative Substitutions II
[074] TABLE 3: Conservative Substitutions III
[075] Exemplary nucleic acids which may be introduced to a host cell include, for example. DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in tire transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, tire term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. Tire type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
[076] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
[077] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g.. Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoom and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Ccnix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinfomiatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of tire siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.
3. Compositions
[078] The invention provides for compositions, which comprise a peptide as described herein, or a nucleic acid encoding the peptide (e.g., an rnRNA or a vector encoding the peptide). Tire composition may be a phannaceutical composition, which comprises the peptide or nucleic acid, and one or more phannaceutically acceptable excipients. A. Peptides
[079] Provided herein are peptides, which may be used in a composition or a method, or for informing the encoding sequence of a nucleic acid. Such peptides are based on those from a discovered repertoire of CNS-derived endogenous regulatory peptides (e.g., guardian peptides), which bind to major histocompatibility complex (MHC) molecules (e.g., MHC class I and MHC class II) where they serve as autoantigens important in autoreactive T cell recognition. During homeostasis, MHC guardian peptides are presented throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes, where they function in the protection against autoimmunity.
[080] The presentation of guardian peptides is diminished in certain disease states, such as neuroinflammatory disease. Administration of a peptide as described herein (e.g., a peptide derived from an MHC guardian peptide) to a subject mediates autoimmunity (e.g., CNS autoimmunity) by supplementing the function of MHC guardian peptides in immune modulation. The peptide may be used for enhancement or expansion of suppressor CD4+ T cell population (e.g., for reduction of CNS autoimmune disease)
[081] The peptide may be derived from an MHC guardian protein that is an MHC I guardian peptide, MHC II guardian peptide, or both. The peptide may be derived from an MHC guardian protein that is CNS-enriched, or an MHC guardian protein that is not CNS-enriched. CNS-enrichment may be elevated expression of the protein in the CNS relative to a control tissue type. In some embodiments, the elevated expression is at least about 2-fold, at least about 3 -fold, at least about 4-fold, or at least about 5 -fold relative to a control. The peptide may be derived from an MHC guardian protein that is a human MHC guardian protein, a murine (e.g., mouse) guardian protein, or both.
[082] In some embodiments, the peptide comprises a fragment of a protein, wherein the fragment of the protein binds to an MHC molecule, hr some embodiments, the protein is expressed at elevated levels in the CNS. In some embodiments, the protein is CNS enriched. In some embodiments, the protein is not expressed at elevated levels in the central nervous system (CNS), or is expressed at decreased levels in the CNS. In some embodiments, the protein is not enriched in the CNS. In some embodiments, the peptide is a fragment of a protein selected from TABLE 4.
[083] TABLE 4: Proteins for fragments that bind to MHC class I and II molecules
[084] In some embodiments, a peptide as described herein is an MHC guardian peptide. In some embodiments, the peptide binds to MHC I, MHC II, or to both. [085] In some embodiments, a peptide comprises or consist of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof. A modified sequence may comprise (e.g., relative to a wild-type MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619), one or more of: oxidation of methionine; deamidation of glutamine; deamidation of asparagine; citrullination of arginine to citrulline; oxidation of cysteine to cysteic acid; or pyroglutamic acid formation from glutamine. In some embodiments, the peptide does not have oxidation of methionine. In some embodiments, the peptide does not have deamidation of glutamine. In some embodiments, the peptide does not have deamidation of asparagine. In some embodiments, the peptide does not have citrullination of arginine to citrulline. In some embodiments, the peptide does not have oxidation of cysteine to cysteic acid. In some embodiments, the peptide does not have pyroglutamic acid formation from glutamine.
[086] A modified sequence may also be a variant sequence (e.g., relative to a wild-type derived MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619). A variant sequence retains its ability to bind to MHC (e.g., MHC I or MHC II). In some embodiments, a variant sequence is at least 80%, at least 85%, at least 90%, or at least 95% identical to a wild-type MHC guardian peptide sequence or to any one of SEQ ID NOS: 1-14619. A variant sequence may comprise one or more amino acid additions or deletions. A variant sequence may comprise one or more conservative amino acid substitutions. In some embodiments, the conservative amino acid substitution is based on a side chain characteristic at the one or more modified positions. In some embodiments, the conservative amino acid substitution is a substitution set forth in TABLE 1 or TABLE 2. In some embodiments, the conservative amino acid substitution is based on the amino acid identity at the one or more modified positions. In some embodiments, the conservative amino acid substitution is a substitution set forth in TABLE 3.
[087] A modified sequence may also be a conjugated sequence (e.g., a wild-type derived MHC guardian peptide sequence or any one of SEQ ID NOS: 1-14619), wherein the peptide is conjugated to a moiety. The moiety may be for enhancing delivery or enhancing targeting of the peptide to the CNS.
[088] TABLES 5-10 categorize the peptides of SEQ ID NOS: 1-14619 based on their CNS enrichment and presence in the MHC peptidome of humans, mouse, or both. This information, along with the protein and start/stop positions from which the peptides derive from, are provided in the remarks of each sequence in the sequence listing accompanying this patent. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 5, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 6, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 8, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 9, or a modified sequence thereof. In some embodiments, a peptide comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 10, or a modified sequence thereof.
[089] TABLE 5: Sequences CNS-Enriched Amino Acid Sequences Identified In Both Human And Mouse Peptidomes
[090] TABLE 6: CNS-Enriched Amino Acid Sequences Identified In Human Peptidome
[091] TABLE 7: Non-CNS-Enriched Amino Acid Sequences Identified In Both Human And Mouse Peptidomes
[092] TABLE 8: Non-Enriched Amino Acid Sequences Identified In Human Peptidome
[093] TABLE 9: CNS-Enriched Amino Acid Sequences Identified In Mouse Peptidome
[094] TABLE 10: Non-Enriched Amino Acid Sequences Identified In Mouse Peptidome
[095] In some embodiments, the peptide is a fragment of a myelin basic protein (MBP). In some embodiments, the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785. 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782. 5783, 5784, 5785, 5786, 5787, 5788, 5789. 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754. 10755, 10756, 10757, 10758. 10759, 10760, 10761, 10762. 10763. 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778,
10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792,
10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806,
10807, 10808, 10809, 10810, 10811, 10812. 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820,
10821, 10822. 10823, 10824, 10825, 10826. 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836. 10837, 10838, 10839, 10840. 10841. 10842, 10843, 10844, 10845. 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546, 14547, 14548, 14549, 14550, 14551, 14552,
14553, 14554, 14555, 14556, 14557, 14558, 14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566,
14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580,
14581, 14582, 14583, 14584, 14585, 14586. 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594,
14595, 14596. 14610, 14611, 14612, and 14613, or a modified sequence thereof. [096] In some embodiments, the peptide is a fragment of a tubulin beta 3 class III (TUBB3) protein. In some embodiments, the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: IN 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, and 14155, or a modified sequence thereof.
[097] In some embodiments, the peptide is a fragment of a neurofilament medium polypeptide (NEFM). In some embodiments, the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:295, 11267, 11268, 11269. 11270, 11271, 11272, 11273, 11274, 11275, 11276, 11277, 11278. 11279, 11280, 11281, 11282. 11283, 11284, and a modified sequence thereof.
[098] In some embodiments, the peptide comprises or consists of the amino acid sequence of, FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
B. Nucleic acids
[099] Provided herein are nucleic acids encoding a peptide as described herein, or a modified sequence thereof. In some embodiments, a nucleic acid encodes a fragment of a protein that binds to MHC (e.g., MHC I or MHC II). In some embodiments, a nucleic acid encodes an MHC guardian peptide. In some embodiments, a nucleic acid encodes an MHC I guardian peptide, an MHC II guardian peptide, or an MHC I/II guardian peptide (i.e., both).
[0100] A nucleic acid may encode for a peptide comprising an MHC -binding fragment of a protein set forth in TABLE 4. A nucleic acid may encode for a peptide comprising or consisting of an amino acid sequence set forth in any one of the SEQ ID NOS set forth in TABLE 5, 6, 7, 8, 9, or 10. A nucleic acid may encode for a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof.
[0101] In some embodiments, a nucleic acid encodes a peptide comprising an MHC-binding fragment of a myelin basic protein (MBP). In some embodiments, the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438. 5439, 5773, 5774, 5775, 5777, 5778, 5779. 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788. 5789. 5790, 5791, 5792, 5793, 5794, 5795, 5796. 10752, 10753, 10754, 10755, 10756. 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770,
10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778, 10779, 10780, 10781, 10782, 10783, 10784,
10785, 10786, 10787, 10788, 10789, 10790, 10791, 10792, 10793, 10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826,
10827, 10828, 10829, 10830, 10831, 10832. 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840,
10841, 10842. 10843, 10844, 10845, 12162. 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544,
14545, 14546. 14547, 14548, 14549, 14550. 14551. 14552, 14553, 14554, 14555. 14556, 14557, 14558,
14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586,
14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, or
14613, or a modified sequence thereof.
[0102] In some embodiments, a nucleic acid encodes a peptide comprising an MHC -binding fragment of a tubulin beta 3 class III (TUBB3) protein. In some embodiments, the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: IN 249, 250, 251, 252, 4829, 14149, 14150, 14151, 14152, 14153, 14154, or 14155, or a modified sequence thereof
[0103] In some embodiments, a nucleic acid encodes a peptide comprising an MHC-binding fragment of a neurofilament medium polypeptide (NEFM). In some embodiments, the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS:295, 11267, 11268, 11269, 11270, 11271, 11272, 11273. 11274, 11275, 11276, 11277, 11278, 11279, 11280, 11281, 11282, 11283. 11284, or a modified sequence thereof.
[0104] In some embodiments, the nucleic acid encodes a peptide comprising or consisting of the amino acid sequence of: FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof.
[0105] Also provided are vectors (e.g., expression vectors) comprising a nucleic acid encoding a peptide as described herein. There may be one or more vectors, wherein each of the one or more vectors comprises at least one or more nucleic acids, as described herein. A vector or multiple vectors may encode multiple different peptides. Such vectors enable production of a transcribable nucleic acid molecule that encodes a peptide as described herein. A vector may further comprise a transcription initiation site, and/or a promoter operably-linked to a transcribable nucleic acid molecule encoding a peptide described herein.
[0106] A vector may be a non-viral vector. In some embodiments, the non-viral vector is a physical vector (e.g., electroporation, sonoporation, magnetofection: or hy droporation). In some embodiments, the non-viral vector is a chemical vector. In some embodiments, the chemical vector is an inorganic particle based, lipid based (e.g., solid or liquid), polymer based, or polypeptide based. In some embodiments, the inorganic particle based vector is selected from calcium phosphate, silica, gold, or magnetic nanoparticle. In some embodiments, the nucleic vector is a nucleic acid/cationic lipid (lipoplex), nucleic acid/cationic polymer (polyplex), or nucleic acid/cationic polymer/cationic lipid (lipopolyplex). In some embodiments, the vector is selected from a cationic lipid, lipid nano emulsion, or solid lipid nanoparticle. In some embodiments, the vector is a polypeptide based vector, a polymer based vector, or a synthetic vector. In some embodiments, the synthetic vector is selected from a polyethylene imine (PEI), chitosal, poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA), dendrimer, polyphosphoester, or polymethacrylate .
[0107] A vector may be a non-viral vector. In some embodiments, the non-viral vector is a physical vector (e.g., electroporation, sonoporation, magnetofection; or hy droporation). In some embodiments, the non-viral vector is a chemical vector. In some embodiments, the chemical vector is an inorganic particle based, lipid based (e.g., solid or liquid), polymer based, or polypeptide based. In some embodiments, the inorganic particle based vector is selected from calcium phosphate, silica, gold, or magnetic nanoparticle. In some embodiments, the nucleic vector is a nucleic acid/cationic lipid (lipoplex), nucleic acid/cationic polymer (polyplex), or nucleic acid/cationic polymer/cationic lipid (lipopolyplex). In some embodiments, the vector is selected from a cationic lipid, lipid nano emulsion, or solid lipid nanoparticle. In some embodiments, the vector is a polypeptide based vector, a polymer based vector, or a synthetic vector. In some embodiments, the synthetic vector is selected from a polyethylene imine (PEI), chitosal. poly (DL- Lactide) (PLA) and Poly ( DL-Lactide- co- glycoside) (PLGA). dendrimer, polyphosphoester, or poly ethacrylate.
[0108] In one example, the nucleic acid may comprise a mRNA encoding a peptide disclosed herein. A nucleic acid as described herein may also be encapsulated. For instance, in some embodiments, a nucleic is encapsulated by a lipid nanoparticle (LNP), polyplex, polymeric nanoparticle, lipopolyplex (LPP), or cationic polypeptide. A nucleic acid may be encapsulated by any particle that is phagocytosed (e.g., for delivery ). A nucleic acid may be encapsulated by any particle that can be up taken by antigen presenting cells.
[0109] A vector may a viral vector. A nucleic acid as described herein by be encapsulated by a viral vector. The viral vectors may? be derived from a virus having CNS or brain tissue specificity (e.g. tropism). In some embodiments, the viral vector is derived from a virus having CNS or brain tissue specificity and not having liver, heart, and/or muscle tropism. The viral vector may be derived from an adenovirus, adeno-associated virus, or retrovirus. In some embodiments, the retrovirus is a lentivirus.
C. Host cells
[0110] Also provided are host cells comprising a nucleic (e.g., a vector or construct) encoding a peptide as described herein. Tire host cells may be used for expression (e.g., production) of a peptide as described herein, or for maintaining a nucleic (e.g., vector or construct) as described herein.. The host cell may or may not be transformed (e.g., transgenic
4. Formulation
[0111] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example. Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such fonnulations will contain a therapeutically effective amount of a peptide as described herein, or a nucleic acid encoding the peptide, which can be in purified form, together with a suitable amount of one or more pharmaceutically acceptable carriers or excipients so as to provide the form for proper administration to the subject.
[0112] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.
[0113] Tire term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Generally, they include substances that are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP/NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF. etc. may also be used.
[0114] The term “pharmaceutically acceptable excipient,” as used herein, can include a solvent, dispersion media, coating, antibacterial and antifungal agent, isotonic, or absorption-delaying agent. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed ), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
[0115] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
[0116] Tire formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to. parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication w itli the agent(s) or attached to tire agent(s) by ionic, covalent. Van der Waals, hydrophobic, hydrophilic, or other physical forces.
[0117] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e.g.. change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
[0118] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.
5. Therapeutic Methods
[0119] Also provided is a method of treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury in a subject in need thereof by administration of a therapeutically effective amount of a peptide as described herein. The peptide may in a composition (e.g., a pharmaceutical composition) as described herein, which is administered to the subject. Also provided are the peptide for use in treating, preventing, or reversing a neuroinflammation- related disorder, an autoimmune neural disorder, or an acute CNS injury; and use of the peptide in the manufacture of a medicament for treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury.
[0120] In some embodiments, the neuroinflammation-related disorder or autoimmune neural disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), acute optic neuritis (AON). Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cold agglutinins, and disialosyl antibodies (CANOMAD), chronic meningitis, Behcet's disease , Central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillar acidic protein (GFAP), Hashimoto’s encephalitis, hypertrophic pachymeningitis, IgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurologic syndrome (PNS), Parkinson’s disease (PD), steroid responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff person syndrome, Susac’s syndrome, and transverse myelitis.
[0121] In some embodiments, the acute CNS injury' is selected from a spinal cory injury, traumatic brain injury’, spinal cord injury’, optic nerve injury, and stroke. [0122] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neuroinflammation-related disorder, autoimmune neural disorder, or an acute CNS injury. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.
[0123] Generally, a safe and effective amount of the peptide (e.g., myelin basic protein (MBP) peptide) is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of the peptide (e.g., MBP peptide) described herein can substantially inhibit a neuroinflammation-related disorder or autoimmune neural disorder, slow the progress of a neuroinflammation-related disorder or autoimmune neural disorder, or limit the development of a neuroinflammation-related disorder or autoimmune neural disorder.
[0124] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. In one aspect, the disclosed composition is infused, injected, or otherwise introduced into the cerebrospinal fluid (CSF) of the subject. In some aspects, the peptide (e.g., MBP peptide) are enclosed in extracellular vesicles or liposomes. The extracellular vesicles may be derived from dendritic cells.
[0125] When used in the treatments described herein, a therapeutically effective amount of the peptide (e.g., MBP peptide) can be employed in pure form or. where such forms exist, in phannaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to prevent, reduce, or reverse a neuroinflammation-related disorder or autoimmune neural disorder.
[0126] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
[0127] Toxicity and therapeutic efficacy of compositions described herein can be detennined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
[0128] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th cd., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw- Hill/ Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single-dose compositions may contain such amounts or submultiplcs thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
[0129] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from the compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
[0130] Administration of the peptide (e.g., MBP peptide) can occur as a single event or over a time course of treatment. For example, meningeal immune cells can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
[0131] Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for prevention, reduction, or reversal of the neuroinflammation-related disorder or autoimmune neural disorder.
[0132] The peptide can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, the peptide can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of peptide, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through the administration of one composition containing two or more peptides, an antibiotic, an anti-inflammatory, or another agent. The peptides (e g., MBP peptides) can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, the peptide can be administered before or after tire administration of an antibiotic, an anti-inflammatory, or another agent.
[0133] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherw ise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed.
6. Administration
[0134] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
[0135] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
[0136] Agents and compositions described herein can be administered in a variety of methods well- known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery' of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
[0137] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
[0138] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve tire transport of tire drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product.
7. Screening
[0139] Also provided are methods for screening. The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
[0140] Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
[0141] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chern Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).
[0142] Candidate molecules for screening according to the methods described herein include both lead- like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug -like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g.. less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Phann. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.
[0143] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being "drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drags within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug -like.
[0144] Several of these "drag-like” characteristics have been summarized into the four rales of Lipinski (generally known as the “rales of fives” because of the prevalence of the number 5 among them). While these rales generally relate to oral absorption and are used to predict the bioavailability of compounds during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drag design efforts such as may be accomplished by using the methods of the present disclosure.
[0145] The four “rules of five” state that a candidate drug -like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drag-like molecules typically have a span (breadth) of between about 8 A to about 15 A.
8. Kits
[0146] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to compositions containing a peptide as described herein (e.g., MBP peptide) optionally enclosed in extracellular vesicles as described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
[0147] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, and sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil- lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.
[0148] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic -readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.
[0149] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed.. Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207- 234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH. ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN- 10: 0954523253). [0150] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
[0151] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Tire recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into tire specification as if it were individually- recited herein. The recitation of discrete values is understood to include ranges between each value.
[0152] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments arc possible without departing from tire scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
EXAMPLES
[0153] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. Those of skill in the art will in light of the present disclosure, appreciate that changes can be made to the specific embodiments described herein and still obtain a like or similar result without departing from the spirit and scope of the invention. All matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense
1. Example 1: Investigating CNS immune privilege - autoantigens fend against CNS autoimmunity
[0154] Experiments were conducted to demonstrate that CNS-derived endogenous peptides promote differentiation of regulatory T cells and modulation of CNS autoimmunity; and that deliver}’ of CNS- derived endogenous peptides broadly safeguards against neuroinflammation-induced pathology.
A. Specific Aims:
[0155] With incidence and prevalence on the rise, autoimmune diseases continue to debilitate countless individuals, increasing healthcare burden while decreasing quality of life. Current interventions, thus far, have focused on non-specific immunosuppressive therapies that come with substantial side effects. To that end. antigen-specific immunotherapies offer an appealing alternative to precisely target organspecific autoimmunity. In an effort to personalize medicine, a deeper understanding of the repertoire of autoantigens presented on major histocompatibility complex (MHC) (e.g.. MHC class I and MHC class II) will direct the development of antigen-specific immunotherapies.
[0156] While autoreactive T cells are well-known for provoking autoimmune disease, their protective roles in limiting tissue damage have been described in heart, skeletal muscle, and central nervous system (CNS) pathologies, including Alzheimer’s disease, stroke, traumatic injury, among others. With evidence supporting a suppressive and beneficial function for autoreactive T cells, the molecular and cellular mechanisms interconnecting the CNS and the peripheral immune system requires further investigation. This search has benefited from the recent rediscovery of a bona fide lymphatic network in the meninges, the membranous coverings that envelop the CNS. Emphasizing a role for the brain borders as an intimate space for interactions between CNS-resident and peripheral immune cells, the brain meninges provide a landscape of opportunities to examine the local presentation of autoantigens to autoreactive T cells. Moreover, meningeal lymphatic vessels serve as conduits for the drainage of antigens and cells to secondary lymphoid organs, specifically the deep cervical lymph nodes (dCLNs). For these reasons, examining CNS-specific autoantigen presentation in CNS associated tissues and how it contributes to immunosurveillance will inform novel mechanisms through which autoantigens maintain tissue homeostasis and protect against overt autoimmunity. [0157] The preliminary' data utilized a specialized platform for immunopeptidomics, spearheading an understanding of the CNS MHC II peptidome. In doing so, an abundance of peptides derived from myelin basic protein (MBP) were identified that centered around a common sequence, MBP33-42, was identified. In exploring a role for these peptides, it was found that in experimental autoimmune encephalomyelitis (EAE), co-immunization of MOG35-55 with our identified MBP peptides ameliorated EAE disease. Further analyses by flow cytometry prior to the peak of EAE disease unveiled an elevated frequency of regulatory Foxp3 -expressing CD4+ T cells in CNS-associated tissues of mice co-immunized with MBP peptides. Moreover, therapeutic delivery' of extracellular vesicles (EVs) packaged with MBP peptides into the cerebrospinal fluid (CSF) could similarly suppress MOG35-55-induced EAE disease when compared to control EVs without MBP peptides. Given these findings, it is hypothesized that the presentation of endogenous CNS autoantigens gives rise to a reserve of regulatory T cells critical for the maintenance and restoration of CNS immune tolerance in both physiology and pathology, respectively.
[0158] Aim 1 is to address a hypothesis that CNS derived endogenous peptides promote differentiation of regulatory T cells, and thus, modulate CNS autoimmunity. Inducing EAE by co-immunizing MOG with MBP peptides resulted in increased frequencies of regulatory T cells. It was hypothesized that MBP specific T cells within the CNS possess regulatory' features that contribute to CNS immune tolerance. To bolster the idea that MBP peptides cultivate Foxp3+ regulatory T cells to ameliorate disease, EAE is induced with MOG and MBP peptides in Foxp3DTR mice with or without diphtheria toxin. Furthermore, the specificity of Foxp3+ regulatory T cells for MBP peptides in the meninges and dCLN is examined utilizing MBP-specific MHC II tetramers.
[0159] Aim 2 is to evaluate a hypothesis that neuropathology limits the presentation of homeostatic epitopes on MHC II molecules. Tissue damage and inflammation lead to epitope spreading, which is thought to further contribute to autoimmune pathology. However, it remains elusive whether the presentation of homeostatic epitopes, such as MBP, are also affected by inflammation. It was expected that neuropathology limits the presentation of MBP peptides, especially those containing the MBP33-42 sequence. MHC II peptidomics of the brain meninges and draining dCLNs was performed at the peak of EAE disease.
[0160] Aim 3 is to test a hypothesis that delivery of CNS derived endogenous peptides broadly safeguards against neuroinflammation -induced pathology. Introducing MBP-containing vesicles into the CSF could suppress EAE disease. Moreover, co-immunization of our MBP peptides with my'elin proteolipid protein (PLP)i39-i5i mitigated EAE disease in SJL/I mice. Thus, it was hypothesized that MBP peptides can be implemented therapeutically to broadly limit neuroinflammation and restore immune tolerance. To demonstrate this, MBP containing EVs were delivered at different time points after the onset of EAE disease. Moreover, EAE was actively induced in different strains, including C57BL/6J, SJL/J, and BIO. PL mice, while delivering MBP-containing vesicles into the CSF to demonstrate broad treatment efficacy of our identified MBP peptides.
[0161] The significance overall of the study was to dissect the functional importance of CNS autoantigen presentation in CNS immunosurveillance. An understanding of this holds potential to artificially induce immune tolerance in other organ systems to prevent allo- and autoimmune attack for clinical benefit.
B. Significance:
[0162] By investigating the CNS antigen repertoire, these studies address barriers that have hampered our capacities to efficaciously employ antigen-specific immune tolerizing therapeutics. Tire first major barrier stems from inadequate knowledge of the repertoire of antigens presented during homeostasis. These studies overcome this barrier by utilizing an optimized technique to unravel native peptides bound to MHC class II (MHC II) molecules in the CNS and its borders. Tire second major barrier arises from the field focusing on a limited number of peptides to investigate autoreactive T cells. Having extensively- characterized the MHC II peptidome in CNS during homeostasis, we now have the ability to broadly understand tire specificities of autoreactive T cells in tire CNS. Moreover, our identified peptides would help direct the development of MHC II tetramers, allowing the tracking and phenotyping of autoreactive CNS-specific T cells. This provides insight into the function of relevant antigen presentation in the CNS to harbor local populations of autoreactive T cells - an understanding of which elucidates mechanisms the CNS employs to acquire immune privilege. Aim Isought to address these two major barriers.
[0163] Another critical barrier to the development of antigen-specific immunotherapies derives from an insufficient understanding of disease-initiating autoantigens and subsequent epitope spreading often arising as a consequence of chronic autoimmune or inflammatory response1’5. These studies overcome this barrier by leveraging our platform technology to define the MHC II peptidome to understand how the repertoire of autoantigens presented on MHC molecules diverges from homeostasis with neuroinflammation. Aim 2 provides a strategy to address this. Along those lines, antigen-specific therapeutic strategies also face a critical barrier of being constrained to identified pathogenic epitopes and finding ways to manipulate either their sequence or their delivery to drive tolerogenic immune responses6’ 10. These studies circumvent this barrier by taking advantage of our knowledge of CNS derived endogenous antigen presentation in homeostasis to inform antigen-specific immunotherapies. Tirus, in contrast to targeting pathogenic autoantigens to directly tolerize against, these studies provide delivery of a therapeutic homeostatic, CNS-specific, MHC Il-bound autoantigen to suppress and protect against CNS autoimmunity. As such, Aim 3 provides a strategy to overcome this barrier.
[0164] These studies provide an expanded comprehension of the repertoire of autoantigens presented and of autoreactive T cells within the CNS. This furthers our understanding of the mechanisms supporting CNS immune privilege and immune tolerance by extension. This fosters an understanding that will guide antigen-specific immunotherapies, enabling the induction of immune tolerance in other tissues to protect against allo- or autoimmune attack for clinical benefit.
C. Background: i) Autoimmune disease:
[0165] Autoimmune diseases remain a major public health concern, incapacitating many individuals with notably increasing rates, ranging from 3-6% annually11 12. In fact, 4.5% of individuals w orldw ide and more than 24 million people in the United States alone are afflicted by a variety of autoimmune disorders13 14. Moreover, with enhanced risks for developing another autoimmune condition15 16, cancers17, and psychiatric disorders18-21, autoimmune diseases negatively impact an individual's quality of life, often leading to lifelong disabilities with undeniable socioeconomic burden22,23. It has been long understood that the major histocompatibility complex (MHC) molecules, also known as human leukocyte antigen (HLA) in humans, play a prominent role in genetic susceptibility to autoimmune disease24-27. These molecules are widely thought to contribute to autoimmune reactivity through the detrimental presentation of autoantigens or foreign antigens that cross react to self-peptides. also referred to as molecular mimicry28-31. As identifying target autoantigens for autoimmune pathogenesis has been challenging, current interventions, thus far, have focused on non-specific immunosuppressive therapies that come with substantial side effects32-36. This suggests a need for strategies that exhibit robust efficacy and tolerability. To that end, antigen-specific immunotherapies offer an appealing alternative to precisely target organ-specific autoimmunity- and has shown promise in different autoimmune conditions37-41. Despite this, antigen-specific approaches have not always been effective and in some cases have been detrimental42-45, exemplifying an incomplete mechanistic understanding of autoantigen presentation in maintaining immune tolerance. ii) Antigen Presentation and T Cell Recognition:
[0166] A series of seminal studies performed during the 1970’s through the 1980’s significantly transformed immunology in our understanding of antigen recognition by T cells. Several lines of evidence began illustrating T cells as an entity possessing the capacity to respond to foreign material but that this response relied on concomitant recognition of self MHC molecules46-48. Subsequent studies would then elucidate a functional role for MHC molecules in presenting peptide antigens to T cells through which structural studies enabled the visualization of peptides ensconced within the binding groove for both MHC I and II molecules49-51. Collectively, these observations improved our understanding of the pivotal interaction between T cell receptors and peptide-MHC complexes in defining the adaptive immune response, inducing tolerance to self while mounting effective immune responses against foreign material.
[0167] Furthermore, MHC molecules readout the state of the cell and the physiology of the tissue at large through shaping the repertoire of antigens presented. Whereas, in the past, antigens were thought to predominantly derive from endogenous sources for MHC I and exogenous sources for MHC II molecules, in recent years, this distinct separation for peptide sources has been blurred. Exogenously derived antigens have been described to funnel through different intracellular pathways to present on MHC I molecules - a process termed cross-presentation52-55. On the other hand, it is also becoming more and more appreciated the abundance of endogenously derived peptides presented on MHC II molecules through which autophagy is thought to play a contributing role56-58. With inflammation, these processes change to accommodate and contextualize the immune response, including enhanced and stabilized surface expression of MHC molecules59-61, modified antigen processing capacities6263, among others. This fosters the focusing of antigen presentation on key molecules to which an immune response is required. Despite this, even in inflammation, endogenously derived peptides still comprise a significant portion of the immunopeptidome64’65. The function of such presentation whether it contributes to the maintenance or breakdown of self-tolerance remains elusive.
Hi) CNS Immune Privilege
[0168] Intriguing early observations noted that certain tissues, such as the CNS, possessed enhanced capacities to tolerize transplantation of skin grafts and heterologous tumors, deeming them immune privileged66-68. However, there remains a significant gap in knowledge with what immune privilege means in the context of systemic immune tolerance, which has largely been explained by central (thymic) and peripheral (non-thymic) mechanisms to remove or functionally inactivate autoreactive T cells69. In fact, this biological need to suppress autoreactive T cells has been contested in the past two decades with studies demonstrating beneficial roles for autoreactive T cells in the heart, skeletal muscles, and CNS, including injury, chronic neurodegenerative conditions, among others70-76. Moreover, where the lack of brain-draining lymphatics explained CNS immune privilege in the past, the rediscovery of a lymphatic network in the meninges, membranous coverings wrapping around the brain, established it as a unique neuroimmune interface77-79. With direct and functional connections to the peripheral immune system, the mechanisms that allow the CNS to achieve immune privilege presents a fascinating framework to reconceptualize immune tolerance. Bringing all of this to bear, whether the repertoire of antigens presented in the CNS contributes to its immune privileged nature remains an important unanswered question. Thus, a goal for these studies to to elucidate mechanisms by which CNS autoantigens entrain autoreactive T cells to sustain and restore immune homeostasis for clinical benefit.
D. Approach
[0169] The These studies enhance our understanding of immune tolerance with potential to guide antigen-specific therapies. Aim 1 builds upon preliminary findings to elucidate regulatory mechanisms of autoantigen dependent protection against autoimmunity. Aim 2 contextualizes the repertoire of autoantigens presented on MHC II molecules in physiology and pathology. Aim 3 advances the therapeutic delivery of autoantigens to reinforce immune tolerance, effectively defending against autoimmune pathology.
E. Preliminary Results
[0170] Knowledge of antigens locally presented on MHC class 11 molecules offers valuable information to expand our understanding of immune tolerance, and this remains largely unexplored. The CNS lending to its immune privileged nature is a fascinating system to explore this. In particular, recent studies described a bona fide brain lymphatic network draining to the deep cervical lymph nodes (dCLNs), linking the CNS to the peripheral immune system77,78. Moreover, it was demonstrated that the meninges, at the brain borders, are a critical site for functional presentation of CNS-derived antigens to patrolling T cells79. As such, the repertoire of antigens presented in the CNS during homeostasis was unknown. MHC II peptidome analysis of the brain meninges and dCLNs of 8-week-old C57BL/6I mice was performed. Gibbs clustering analysis revealed the I-Ab binding motif and was consistent with prior studies (FIG. 5A)80-81. We then analyzed the total list of MHC-II-bound peptides in the dCLNs and brain meninges for proteins with elevated expression in the CNS when compared to other tissues82; 6.28% and 17.44% of MHC-II-bound peptides in the dCLNs and brain meninges, respectively, derived from CNS elevated proteins (FIG. 5B). Intriguingly, MBP-derived peptides were prominently presented in tire brain meninges, comprising 77% of peptides that could be defined as CNS elevated (FIG. 5B). The abundance of MBP presentation in the brain meninges was surprising and elucidation of its role in CNS immune tolerance was sought. Moreover, when aligning identified MHC-II-bound peptides to tire MBP sequence, it was found that a vast majority of MBP-derived peptides centered around a common amino acid sequence, MBP33-42. To investigate this. MBP peptides that were identified in our MHC II peptidome were synthesized, which either included or excluded the common MBP33-42 sequence. ii) Endogenous peptide suppresses myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)
[0171] Given the preponderance of MBP presentation in the brain meninges, we wondered whether our identified MBP peptides from the CNS MHC II peptidome could induce or modulate the course of CNS autoimmune disease. To answer this question, the EAE an animal model for multiple sclerosis (MS) was utilized. 8-week-old C57BL/6J mice were immunized with MOG35 55 alone, as our positive control. MBP12.26 (i.e., KYLATASTMDHARHG (SEQ ID NO: 14612)), or MBP2742. Mice were tracked by standard EAE scoring for 20 days post-immunization (dpi) (FIG. 6A). The control group immunized with MOG35-55 exhibited hindlimb motor symptoms between 9 and 12 dpi reaching peak severity between 14 and 18 dpi, consistent with prior studies83. This was in stark contrast to the group immunized with cither MBP12-26 or MBP27-42 with which 0 of 5 mice displayed neither clinical nor pathological signs of EAE (FIG. 6B). These findings were in line with prior observations that the C57BL/6J strain is particularly resistant to MBP-induced EAE84,8’. Despite the inability of immunizing MBP peptides alone to induce pathology, it was contemplated whether MBP peptides could modulate the disease course of MOG35 55- induced EAE. To address this question, 8-week-old C57BL/6J mice were co-immunized with MOG35-55 and MBP12-26, MBP2742, or MBP27-42(citrullinatcd). Intriguingly, where MBP27-42 co-immunization with MOG35-55 exhibited statistically significant suppression of the EAE phenotype, neither MBP12-26 nor MBP27-42(citrullinated) co-immunization attenuated the disease, exhibiting similar outcomes as the positive control (FIG. 7A). Moreover, the peak EAE score was significantly decreased for the MOG35-55 + MBP2742 group when compared to all other treatment modalities (FIG. 7B). Accordingly, this finding highlighted that the observed suppression of MOGss-ss-induced EAE with MBP27-42 did not simply occur as a result of co-immunizing an additional peptide - the co-immunization of MBP12-26, an autoantigen of similar length, failed to suppress disease. But even more than that, the experiment was controlled by directly modifying the MBP2742 sequence with citrullination, a disease relevant enzymatic modification. In fact, peptidyl arginine deiminase (PAD), a family of enzymes catalyzing the conversion of arginine to citrulline residues, becomes dysfunctional in a number of autoimmune disease, including MS80,87. Prior studies have also demonstrated that MBP itself becomes hypercitrullinated in MS and EAE and that this correlates with increased disease severity87,88. Fascinatingly, co-immunization of MOG35-55 with citrullinated MBP2742 abrogated the capacity of MBP2? 42 to mitigate EAE disease (FIGS. 7A-7B).
Despite the hypothesis that citrullination elicits neoepitopes triggering autoreactivity, a recent study rather described an absence of T cell responsivity to citrullinated myelin-associated peptides89. Contextualizing this with our finding, it is foreseeable that the citrullination of autoantigens rather hinders regulatory immune responses, releasing the brakes set in place to resist autoimmunity. These preliminary findings bolster a specific immunomodulatory function for MBP27-42, motivating a directed evaluation of its binding epitope to better understand the T cell response it generates. Experiments for achieving this are described in Aim 1.
Hi) Co-immunization with a MBP peptide skews the quality of T cell response towards a regulatory phenotype in MOG-induced EAE
[0172] The stark decrease in clinical EAE severity with MBP2742 co-immunization with MOG35-55 brought to question whether MBP2742 peptides regulated T cell response in EAE. Initial exploration of this was performed by assessing changes to T cell differentiation, focusing on the expression of transcription factors. This evaluation was predicated on the establishment of a critical role for RORyt+ Thl7 cells in the pathogenesis of EAE90. Moreover, a plethora of studies demonstrated the capacity of Foxp3 to suppress T cell effector function and more specifically to antagonize the function of RORyt91'94. It was hypothesized that MBP2742 regulated the pathogenicity of MOG^ ^-induccd EAE by modulating the expression or functionality of RORyt. To test this, 8-week-old C57BL/6J mice were immunized with either MOG35-55 or MOG35-55 + MBP27-42 to induce EAE. dCLNs were harvested at day 13 and the brain meninges at day 16 post EAE induction and flow cytometry was perfonned to assess for any changes to T cells. Prior to the peak of EAE, the dCLNs of co-immunized MOG35-55 + MBP2742 mice already reflected a statistically significant increase in frequency of Foxp3+ CD4+ T cells with a concomitant downward trend noted to the frequency of RORyt+ expression when compared to MOG35-55 (FIGS. 8A-8B). This paralleled observations in the brain meninges at the peak of EAE disease. There, the proportion of CD4+ T cells expressing RORyt was significantly decreased in tire MOG35-55 + MBP27-42 group with an accompanying upward trend in the frequency of Foxp3+ CD4+ T cells (FIGS. 8C-8D). Altogether, our preliminary results call attention to the notable fortification of regulatory Foxp3+ CD4+ T cells in the dCLNs followed by the diminished presence of pathogenic RORyt+ CD4+ T cells in the brain meninges at the peak of disease. This provides evidence for MBP27-42 in regulating pathogenic T cell responses in EAE that may be mediated by Foxp3, which is delineated in Aim 1. iv) Co-immunization with a MBP peptide suppresses EAE in a different mice strain
[0173] Intriguingly, MHC II peptidome analysis of the brain meninges of SJL/J mice revealed an abundant presentation of MBP peptides that similarly contained the MBP33-42 sequence. Curious as to whether MBP27-42-mediated protection against neuroinflammation could be generalizable, we utilized another active EAE model with SJL/J mice. Here, we immunized 8-week-old SJL/J male mice with myelin proteolipid protein (PLPjug 151 or co-immunized with either MBP27-42 or neurofilament light polypeptide (NF-L)ieo-i73 to induce EAE disease. NF-L160-173, an endogenous peptide of similar length to MBP27-42, was selected as an appropriate control peptide with evidence supporting its presentation on MHC II molecules in EAE with limited T cell reactivity in SJL/J micegs.ge. Moreover, NF-L160-173 was identified as an MHC II -bound peptide in the brain meninges of SJL/J mice. Consistent with literature, immunization of PLP139-151 in young male SJL/J mice exhibited monophasic EAE disease97. To our surprise, co-immunization with MBP2742 but not NF-Li60-i73 demonstrated significant suppression of EAE with a notable downward trend to peak EAE scores (FIGS. 9A-9B). Similar to what was observed in C57BL/6J mice, the co-immunization of another peptide, NF-L160-173, with PLP139-151 did not explain the suppressive effects observed when co-immunizing with MBP2742 Collectively, this observation not only further supports a specific immunomodulatory function for MBP27-42 peptides but also posits a potentially generalized role for MBP peptides in fending broadly against CNS autoimmunity. v) Therapeutic delivery of MBP peptides in extracellular vesicles (EVs) mitigates CNS autoimmune damage
[0174] Hie past two decades have exhibited an increasing appreciation for EVs in regulating immune responses98. From loading nucleic acids to small -molecule drugs, EVs have considerable immunotherapeutic potential99 100. A recent study showed that EVs from oligodendrocytes could suppress autoimmune neuroinflammation101. Following this therapeutic angle, EVs were isolated from dendritic cells using ultracentrifugation and enrichment was confirmed both by immunoblot and transmission electron microscopy (FIGS. 10A-10B). MBP 27-42 peptides were introduced via sonication into the E s following established protocols100 102. Intriguingly, the injection of MBP27-42 containing EVs into the CSF on day 5 post induction of EAE by MOG35 55 immunization significantly dampened clinical EAE scores when compared to injection of EVs lacking MBP2742 (FIGS 10C-10D). This finding demonstrates the exciting immunotherapeutic potential of MBP peptides as explored in Aim 3.
F. Experimental Design: [0175] Age-matched mice were used in the experiments. If mice in the experimental group were obtained from an outside provider, so were the respective controls. Transgenic mice and their wild-type littermate controls were bred in the same facility. Assessment was made using both males and females. For in vivo studies, 10 animals per group were utilized based on the following statistical criteria: the expected difference is about 50% with SD 0.33, double-sided, alpha power 0.9, p-value 0.05: N = 10. For imaging and flow cytometry analysis, 5 animals per group were utilized based on the following statistical criteria: the expected difference is about 37% with SD 0.20 for each, double-sided, alpha power 0.8, p- value 0.05: N=5. To enhance rigor, all measurements were performed with investigator blinded to group identity; group identity’ is only revealed after completion of data recording. i) Aim 1: CNS derived endogenous peptides promotes differentiation of regulatory T cells, and modulate CNS autoimmunity
[0176] Preliminary studies indicate that the presentation of endogenous peptides regulates T cell response to dampen neuroinflammation in EAE. However, whether MBP peptides gamer a population of regulatory T cells remains unclear. By specifically ablating Foxp3+ regulatory T cells as well as utilizing tetramers to phenotypically assess MBP specific T cells, the regulatory mechanisms imparted by endogenous peptide presentation to protect the CNS from overt autoimmunity can be determined.
Study #1: Ablation of Foxp3 to evaluate MBP^^-dciicndent regulation of T cell response
[0177] Preliminary results reveal that co-immunization of MOG35-55 + MBP2742 suppresses EAE with a notable shift in ratio between RORyt and Foxp3 expression on CD4+ T cells. To confinn a role for MBP27-42 in cultivating a population of Foxp3+ regulatory T cells. 8-week-old Foxp3DTR transgenic mice are utilized, which express a diphtheria toxin receptor (DTR) under the Foxp3 promoter. Upon treating Foxp3DTR mice yvith diphtheria toxin, Foxp3+ T cells can be selectively ablated. As our control, 8- yveek-old Foxp3EGFP mice are utilized, yvhich only express enhanced green fluorescent protein (EGFP) under the control of the Foxp3 promoter. EAE is induced in both transgenic mice with either MOG35-55 alone or MOG35 55 + MBP27-42. Diphtheria toxin is injected intraperitoneally and intravenously (200 ng) on day 3 post EAE onset. All experimental groups are subjected to standard EAE scoring for up to 21 days. Tissues are harvested, including brain meninges, at day 16, the peak of EAE disease, for analysis by both floyv cytometry and immunohistochemistry. By using Foxp3EGFP control mice, recapitulate of preliminary findings is expected, demonstrating an upward trend to Foxp3 expression in the brain meninges following co-immunization of MOG35-55 + MBP27-42 when compared to MOG35-55. Specific ablation of Foxp3+ regulatory T cells in Foxp3DTR mice is expected to abrogate MBP2742 dependent amelioration ofEAE, exhibiting a mechanism that relies on Foxp3 -mediated suppression.
Study #2 Determining the role of MBP27-42 specific CD4+ T cells in regulating CNS immune tolerance
[0178] Preliminary data demonstrates that MBP2742 is presented on MHC class II molecules in the CNS during homeostasis and that MBP27-42 affords protection against MOG3555 -induced EAE. To delineate the function of MBP27-42 specific CD4+ T cells, MHC II tetramers were recently developed using the shared amino acid sequence, MBP3342. These tetramers are tested and calibrated using lymphocytes isolated from the C57BL/6J mice immunized with MBP27-42 peptides boosted with complete Freund’s adjuvant (CFA) bilaterally in flank regions. One week is allowed for generation of a sufficient immune response prior to isolation of the draining inguinal lymph nodes. In tandem, the isolated T cells are fused with BW5147, a thymic lymphoma cell line, to generate T cell hybridoma clones. In vitro antigen presentation assays are performed by co-culturing individual T cell clones with M12.C3, a B lymphoma cell line serving as the antigen presenting cell, in the presence of MBP2742 Using IL-2 enzyme linked immunosorbent assay (ELISA) as our readout for T cell activation, T cell clones specific to MBP2742 are selected. With guidance by MHC II binding affinity algorithms, key amino acids of MBP2742 are mutated to identify residues that abrogate activation of MBP2742 specific T cell clones. By discerning critical recognition events for peptide-MHC II and T cell receptor interaction, the development of MBP27-42 specific MHC II tetramers can be better directed. Using our developed tetramers, MBP2742 specific T cells are phenotyped in the brain meninges and dCLN by flow cytometry of wild-type mice, mice immunized with MBP2742 alone, and mice co-immunized with MOG35-55 + MBP2742. Cell surface markers and transcription factors, including Foxp3, are stained. In doing so, whether the CNS harbors a population of MBP27-42 specific CD4+ T cells that express markers of regulation, such as Foxp3, contributing to CNS immune tolerance, can be detennined.
[0179] Collectively, the experiments described in Aim 1 determine the role of endogenous peptide presentation in modulating T cell response, while exploring Foxp3-mediated regulation as a mechanistic basis for this phenotype.
[0180] Based on preliminary results, it is hypothesized that MBP27-42 recruits a reservoir of Foxp3+ rcgulatory T cells for the upkeep of CNS immune tolerance. However potentialy, MBP27-42 specific T cells may enforce immune regulation through other regulatory mechanisms. For example, prior studies have demonstrated a role for Foxp3- T regulatory type 1 (TRI) cells in regulating autoimmunity, including EAE, through production of regulatory cytokines, including IL-10 and TGFP103.104. To that end, intracellular cytokine staining for IL- 10 and TGFp is included when phenotyping MBP2742 specific T cells by flow cytometry. Additionally, IL- 1 Ofl/fl and CD4-CreERT2 are both commercially available and permit conditional deletion of IL-10 production in CD4+ T cells. ii) Aim 2: Neuropathology limits the presentation of homeostatic epitopes on MHC II molecules.
[0181] Lending to inflammation induced tissue damage, the spreading of epitopes and its contribution to autoimmune pathology has been described, including in EAE1'5. However, the impact of epitope spreading and inflammation on the autoantigenic repertoire in the CNS is unknown. Further, its impact on homeostatic presentation of MHC -II bound peptides remains elusive. To assess any changes to meningeal lymphatic drainage, fluorescently conjugated ovalbumin protein was introduced into the CSF via intracistema magna (i.c.m.) injection at the peak of EAE disease and assessed the draining dCLNs. In doing so, statistically significant increase in the ovalbumin coverage in the dCLNs of EAE mice was found, when compared to CFA controls (FIGS. 11A-11C). Moreover, a prior study demonstrated that ligation of afferent lymphatics into the dCLN could mitigate EAE disease, exhibiting its relevance in EAE disease progression78. As such, in this aim a broad survey the immunopeptidome landscape is sought for assessment of the divergence in the CNS MHC II peptidome between inflammation and homeostasis with the expectation that the presentation of our identified MBP peptides decline.
Study #1: Establishment of the CNS MHC II peptidome during neuroinflammation
[0182] Preliminary data reveals a notable increase in meningeal lymphatic drainage at the peak of EAE disease warranting investigation into the autoantigenic repertoire presented in the brain meninges and its draining peripheral lymph node, dCLNs. To address this, C57BL/6J mice are immunized with either CFA alone or CFA + MOG3555; pertussis toxin is administered on day of immunization and two days later. All experimental groups are subjected to standard EAE scoring for 16 days after which tissues, including brain meninges and dCLNs are harvested and processed. Hie individual tissues are lysed with mild detergents in the presence of protease inhibitors to preserve the peptide-MHC complexes. Hie lysate pre-cleared of debris by centrifugation will then be incubated with sepharose conjugated to an antibody specific to the MHC class II molecule. Following a series of wash steps, peptides are eluted from MHC class II molecules with 10% acetic acid. Eluted peptides are then cleaned further with C 18 spin columns prior to being subjected to mass spectrometry and immunological analyses. By establishing the MHC II peptidome during active EAE disease, the presentation of MBP peptides, particularly those containing the common shared sequence, MBP33-42, is expected to diminish as a result of epitope spreading and diversification.
[0183] The experiments of Aim 2 seek to unveil alterations to the autoantigen repertoire, specifically in the CNS, when comparing between homeostasis and inflammatory disease. In doing so, the study demonstrates diminished presentation of homeostatic autoantigens that play a pivotal role in maintenance of CNS immune tolerance.
[0184] Potentially, immunization of CFA in the absence of MOG35-55 along with the administration of two doses of pertussis toxin may induce changes to CNS border tissues that confound differences specific to EAE disease. Taking this into consideration, naive C57BL/6J mice are included as another control when harvesting tissues, and they are subjected to the same pipeline for immunopeptidomics. Additionally, given that the EAE disease course in C57BL/6J mice is monophasic, it is possible that the autoantigenic repertoire at the peak of EAE disease is already stabilized as it enters the chronic phase of disease. To address this, harvests are staggered to collect for brain meninges and dCLNs both before peak of disease (day 10) and after peak of disease (day 21). This further leverages the immunopeptidomics platform in the CNS to provide a more comprehensive picture regarding the dynamics of EAE disease on the MHC II peptidome of CNS-associatcd tissues over time.
Hi) Aim 3: Therapeutic delivery of CNS derived endogenous peptides broadly safeguards against neuroinflammation-induced pathology
[0185] Preliminary results suggest a potentially generalizable role for MBP derived peptides in regulating neuroinflammation, making it an attractive therapeutic target. Co-immunization of MBP27-42 peptides with PLP139-151 in SJL/J mice, a different active EAE model, significantly mitigated disease progression. Moreover, injection of EVs containing MBP2742 into the CSF prior to the onset of paralytic symptoms attenuated the extent of EAE disease. Both observations highlight promising immunotherapeutic potential for MBP peptides. Additionally, the targeting properties and high biocompatibility of EVs provides a modifiable carrier to further fine-tune antigen-specific immunotherapies. It is examined whether the delivery of MBP27-42-containing vesicles into the CSF ameliorates pathology even when administered after the onset of paralytic symptoms and whether it broadly protects against CNS autoimmunity in different mice strains.
Study #1: Evaluating peptide contents of EVs free-floating in the CSF during homeostasis [0186] A recent study in humans demonstrated that myelin-associated proteins, including MOG, PLP, and MBP, could be found within EVs derived from both serum and CSF of both healthy and patients affected by MS105. Moreover, with preliminary data suggesting a beneficial function for MBP peptides encapsulated in EVs, the content within EVs present in the CSF during homeostasis requires investigation. To assess this, CSF is collected using glass micro-capillary pipette from naive C57BL/6J mice. Using the Exo-spinTM system, EVs are precipitated and subjected through size exclusion chromatography to purify for EVs specifically. Confirmation is by immunoblot and ZetaView nanoparticle tracking analyzer prior to further downstream application. Following confinnation, exosomes undergo at least five freeze-thaw cycles to release intracellular contents; which is purified by C18 columns enriching for peptides. The collected material is subjected to mass spectrometry and immunological analyses.
Study #2: Assessing the therapeutic capacity of EVs containing MBP27-42 peptides to treat neuroinflammation
[0187] Preliminary data supports a beneficial function for vesicle-mediated delivery of MBP2742 peptides when injected into tire CSF prior to the emergence of any EAE symptoms. However, its capacity to curb ncuroinflammation after tire onset of paralysis remains unexplored. To explore this, EVs arc enriched from DC2.4 cells, an immortalized dendritic cell line, with 100,000xg ultracentrifugation and confinn purity by immunoblot, TEM, and ZetaView nanoparticle tracking analyzer. Enriched vesicles are sonicated equally in the absence or presence of endogenous MBP27 42 and encephalitogenic MOG35-55 peptides. Vesicles are further purified by both size exclusion chromatography and high-speed ultracentrifugation to remove any unincorporated peptides, effectively isolating intact control vesicles or vesicles containing our peptides of interest. Quality of enriched EVs is assessed similarly as mentioned above and further quantified by ZetaView nanoparticle tracking analyzer to control for the total number of vesicles injected into the CSF. Moreover, loaded peptides are fluorochrome conjugated enabling control for loading efficiency by nanoparticle flow cytometry. EAE is induced with MOG35-55 immunization in 8-week-old C57BL/6J mice. Subsequently, control vesicles or vesicles containing either MBP27-42 or MOG35-55 are introduced into the CSF via intracistema magna (i.e.m.) injection on day 5 (prior to disease onset), day 10 (onset of paralytic symptoms), or day 16 (peak disease). All experimental groups with their respective littermate controls are subjected to standard EAE scoring for up to 25 days. In doing so, only the delivery of MBP27-42-containing vesicles across different time points is expected to exhibit therapeutic benefit, promoting the restoration of CNS immune tolerance. Furthermore, intravenous injection of EVs are also examined as it presents with a minimally invasive and convenient mode of delivery.
Study #3: Elucidating a function for endogenous MBP27-42 peptides in broadly suppressing CNS autoimmunity
[0188] Preliminary findings substantiate a more general immunomodulatory role for MBP27-42, an epitope abundantly presented on MHC II molecules, in regulating CNS immune homeostasis. In a completely different strain of mice with a different peptide to actively induce EAE, MBP2742 sufficiently suppressed against PLPw-isi-induced EAE in SJL/J mice. Moreover, the MBP27-42 sequence itself is highly conserved across different vertebrate species, including humans106 107. For these reasons, interrogation of the capability of MBP27-42-containing vesicles to dampen neuroinflammation-induced damage in different active EAE models is sought. The established pipeline as described above in Study #1 is followed to enrich for control vesicles and vesicles containing either MBP27-42 or appropriate control peptides. Active EAE is induced through immunization of MOG35-55, PLP139-151, or N-terminally acetylated MBPMI in C57BL/6J, SJL/J, or B10.PL mice, respectively. Soon thereafter, mice receive i.c.m. injections of control vesicles or vesicles containing peptides on day 5 after which they are subjected to standard EAE scoring for up to 25 days. Given that both co-immunization of MBP27-42 with PLP139 151 in SJL/J mice and delivery of MBP27-42-containing vesicles into the CSF of MOG35-55-immunized C57BL/6J mice sufficiently suppressed EAE disease, therapeutic delivery of MBP27-42-containing vesicles is expected to similarly protect against different active EAE models.
[0189] Collectively, the experiments described in Aim 3 unravel a broad immunomodulatory function for endogenous MBP2742 peptides in defending against CNS autoimmunity, demonstrating its immunotherapeutic potential to control neuroinflammation-induced damage and restore CNS immune tolerance.
[0190] Based on preliminary results, MBP peptides are predicted to exhibit therapeutic efficacy at different stages of disease and in different strains of mice. However, the observed suppression of EAE disease may remain restricted to i.c.m. delivery of MBP2742 at day 5, prior to the onset of paralysis. As EVs reflect the cell type and environment from which they are derived108 109, modulating the character of vesicles may bolster our antigen-specific immunotherapeutic approach. A recent study demonstrated that vesicles derived from dendritic cell cultures treated with low dose IFNy reduced oxidative stress and enhanced CNS myelination processes in vivo110. Moreover, EVs derived from mesenchymal stem cells mildly but significantly reduced clinical EAE scores when administered intravenously at the peak of disease11 this strengthens confidence in treating CNS autoimmunity according to the invention at a more advanced stage of disease. Thus, by harnessing the modulatory capacity of EVs with the antigenspecificity of the approach described herein, MBP27-42 and control peptides are also loaded into vesicles derived from IFNy-stimulated dendritic cells and mesenchymal stem cells. In conclusion, these studies further advance MBP27-42-containing vesicles as an antigen-specific immunotherapeutic for autoimmune disease.
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Tri cell-dependent active tolerance blunts the pathogenic effects of determinant spreading. J Clin Invest 110, 701-710 (2002). https://doi.org: 10. 1172/JCI15176. Clemente-Casares, X. et al. Expanding antigen-specific regulatory' networks to treat autoimmunity. Nature 530, 434-440 (2016). https://doi.org: 10.1038/nature 16962 . Galazka, G., Mycko, M. P., Selmaj, L, Raine, C. S. & Selmaj. K. W. Multiple sclerosis: Serum- derived exosomes express myelin proteins. Mult Scler 24. 449-458 (2018). https://doi.org: 10. 1177/1352458517696597 . Streicher, R. & Stoffel, W. The organization of the human myelin basic protein gene. Comparison with the mouse gene. Biol Chem Hoppe Seyler 370, 503-510 (1989). https://doi.org: 10. 1515/bchm3.1989.370. 1.503 . Stoner, G. L. Conservation throughout vertebrate evolution of the predicted beta-strands in myelin basic protein. J Neurochem 55, 1404-1411 ( 1990). https://doi.org: 10. 1111/j .1471- 4159.1990.tb03153.x . Harting, M. T. et al. Inflammation-Stimulated Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Inflammation. Stem Cells 36, 79-90 (2018). https://doi.org: 10.1002/stem.2730. Ojeda-Hernandez, D. D. et al. Exosomes and Biomaterials: In Search of a New Therapeutic Strate-gy for Multiple Sclerosis. Life (Basel) 12 (2022). https://doi.org: 10.3390/life 12091417. Pusic. A. D., Pusic, K. M., Clayton. B. L. & Kraig, R. P. IFNgamma-stimulated dendritic cell exosomes as a potential therapeutic for remyelination. J Neuroimmunol 266, 12-23 (2014). https://doi.org: 10.1016/j.jneuroim.2013.10.014 . Riazifar, M. et al. Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neuro- degenerative Disorders. ACS Nano 13, 6670-6688 (2019). https://doi.org: 10.1021/acsnano.9b01004 2. Example 2: Identification and purification of MHC-binding peptides that modulate neuroinflammation
[0191] Experimental methods for the identification and purification of peptides that modulate neuroinflammation are provided, along with results.
A. Background:
[0192] A series of seminal studies performed during the 1970’s through the 1980’s transformed immunology’s understanding of the antigen specificity of T cells. In fact, several lines of evidence demonstrated T cells as an entity possessing the capacity to respond to foreign material but that this response relied on concomitant recognition of major histocompatibility complex (MHC) molecules. Subsequent studies elucidated a functional role for MHC molecules in presenting peptide antigens to T cells through which structural studies enabled the visualization of peptides ensconced within the binding groove for both MHC I and II molecules. Despite sharing a similar structure, the requirements for peptide binding by either MHC I or II molecules result in starkly different binding motifs; this obviates preference for amino acids at certain positions along the peptide binding groove (FIGS. 12A-12C).
[0193] MHC I molecules place tyrosine residues at the end of the peptide binding pocket, often restricting the peptide length to 8-10 amino acid residues, although longer peptides bound to MHC I molecules have been reported. In contrast, the MHC II molecules are more open at the ends allowing for peptides to extrude beyond the confines of the binding cleft. As such, the MHC II molecules are more amenable and accommodate peptide lengths of around 15-25 amino acid residues. Moreover, the portions of the peptide not directly involved with the peptide binding groove of the MHC II molecule have been posited to play a role in influencing the stability of peptide binding. This adds an extra element of peptide association for MHC II molecules that of which is currently thought to be absent for MHC I molecules.
[0194] Beyond differential requirements for peptide binding between MHC I and II molecules, the source and processing machinery for antigens have also been thought to shape the flavor of antigens presented by either MHC I or II molecules. In fact, in the past, antigens were thought to predominantly derive from endogenous sources for MHC I and exogenous sources for MHC II molecules. However, in recent years, this distinct separation for peptide sources has rather been blurred. In fact, exogenously derived antigens have been described to funnel through different intracellular pathways to present on MHC I molecules - a process termed cross-presentation. On the other hand, it is also becoming more and more appreciated the abundance of endogenously derived peptides presented on MHC II molecules through which autophagy is thought to play a contributing role. With inflammation, these processes, of course, change to accommodate and contextualize the immune response, including enhanced and stabilized surface expression of MHC molecules, modified antigen processing capacities, among others. This fosters the focusing of antigen presentation on key molecules to which an immune response is required. Despite this, even in inflammation, endogenously derived peptides still comprise a significant portion of the immunopeptidome. The function of such presentation and whether it contributes to the maintenance or breakdown of self-tolerance remains elusive. In order to tackle this, here, we establish the MHC I and II peptidomes in both mice and humans and evaluate shifts to the autoantigen repertoire under different conditions.
B. Protocol:
[0195] Harvest and process tissues following established protocols to acquire cell pellet per each sample. The downstream processing of samples after this step may follow as below. i) Day 1 (Immunopurification of MHC I or MHC II)
Prior to lysing, optionally wash the cell pellet (e.g., once with PBS buffer).
1. Spin down cells (e.g., 1,000g for 5 mins).
2. Aspirate and add 1 mL/sample of lysis buffer (e.g., MEGA8/9 lysis buffer) plus one or more protease inhibitors (e.g., Roche protease inhibitor cocktail, PMSF, lodoacetamide, Leupeptin). Pipet up and down to thoroughly suspend cell pellet.
3. Lyse cells (e.g., for 1 hour in cold room while rocking).
4. Centrifuge the lysate (e.g., 20.000g for 25 mins at 4°C) a. Debris/nuclei/etc. will be pelleted, leaving protein of interest in the supernatant.
5. Select supernatant (avoid touching the pellet) and apply to purification media (e.g. IgG Sepharose).
6. Rock at 4°C for 30 mins.
7. Spin down the sample (e.g., 500g for 5 mins).
8. Select supernatant and apply onto specific MHC antibody conjugated to the sepharose resin. Particular information for MHC antibodies are provided below. Rock in the cold room overnight. a. Human MHC I (pan HLA-A/B/C) = Clone W6/32 b. Human MHC II (HLA-DR) = Clone L243 c. Human MHC II (HLA-DQ) = Clone la3 d. Mouse MHC I (H2-Kb ) = Clone Y3 e. Mouse MHC I (H2-Db ) = Clone B22/249 f. Mouse MHC II (H2-Ab ) = Clone Y3-P ii) Day 2 (Column Purification)
[0196] 1. Apply sample from Day 1 to chromatography column (e.g., BioRad column).
2. Wash samples (e.g., as follows: a. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4; b. 5-10 mL 400 mM NaCl 20 mM Tris pH 7.4; c. 5-10 mL 150 mM NaCl 20 mM Tris pH 7.4: d. 5-10 mL 20 mM Tris pH 8.0)
3. Elute peptides off the MHC I- or MHC Il-conjugated Sepharose (e.g., with 10% acetic acid).
4. Freeze samples (e.g., -20C).
5. Apply vacuum to the samples (e.g. SpeedVac) to dry the peptides down (about 6 hours). a. At completion, a pellet is present (e.g., chalky white, or sometimes gelatinous-like)
Hi) Day 3 (Optional Peptide Clean-up):
[0197] At this point (by day 2), peptides bound to MHC I or MHC II molecules have been purified, but there may be residual detergents or smaller interfering molecules, which may be removed. Steps include:
Detergent Removal Column (Pierce) (follow manufacturer's protocol, briefly):
1. Spin down the storage buffer of which the column is initially kept (e.g.. 1 ,500g/ 1 min).
2. Wash the column 2-3 times with 20mM Tris pH 8 to make the column ready for use.
3. Resuspend each samples in buffer (e.g., ~20 mM Tris pH )8 and apply to the resin.
4. Spin down (e.g., l,500g/2 mins). a. The resin will bind the detergents allowing peptides to flow through.
C18 Spin Column (follow manufacturer’s protocol, briefly):
1. Supplement the eluted sample from step 4 (above) with 20% acetonitrile/2% TFA solution. Keep samples on icc.
2. Activate Cl 8 spin columns with 50% acetonitrile solution.
3. Equilibrate the C18 spin column with a solution containing 5% acetonitrile/0.5% TFA.
4. Load samples from ice onto the C18 spin column, spin down (e.g., l,500g/l min).
5. Optionally reapply flowthrough to the Cl 8 spin column to maximize binding of peptides resin.
6. Wash the column with solution containing 5% acetonitrile/0.5% TFA. 7. Elute the sample with a 95% acetonitrile solution. Spin down (e g., 1 ,500g/l min).
8. Freeze the samples on dry ice (5-10 mins).
9. Apply vacuum to the samples (e.g. SpeedVac) to dry peptides down (about 4 hours).
[0198] Samples can analyzed using LC-MS/MS for identification of peptidome.
3. Example 3: Analyzing MHC I and II peptidomes in both humans and mice
[0199] The peptidomes of antigens presented by MHC molecules in brain and lymphatic tissues of humans and mice were investigated by purification and identification using LC/MS. 14,609 peptides were identified in a protcomic assay of autoantigens presented on major histocompatibility complex classes I and II (MHC I and MHC II) in human and mouse and peripheral and CNS immune cells (i.e., SEQ ID NOS: 1-14,609). Peptides were analyzed for the following properties: a. whether shared by both humans and mice, or in humans only, or in mice only; b. binding to MHC I, to MHC II, or to both; c. enrichment in the CNS; and d. identification of the protein from which the peptide derives from, including beginning and end amino acids for the peptide (fragment) of the protein.
[0200] The resulting sequences of the peptides identified in the MHC I and MHC II peptidome are provided for in the ST.26 formatted sequence listing accompanying this patent (see specifically SEQ ID NOS: 1-14619). The analyzed properties for each of the peptides is presented in the sequence listing (i.e., at the remarks for each of SEQ ID NOS: 1-14619), including information on which proteins the identified peptides derive from, the starting and ending positions for each peptide (fragment of the protein), their human/mouse specificity, and their specificity for MHC I, II, or both.
4. Example 4: MHC-II peptidome of the CNS reveals endogenous regulatory (guardian) peptides
A. Abstract:
[0201] The central nervous system (CNS), despite the presence of strategically positioned anatomical barriers designed to protect it, is not entirely isolated from the immune system. In fact, it remains physically connected to and can be influenced by the peripheral immune system. How the CNS retains such responsiveness while maintaining an immunologically unique status remains an outstanding conundrum. In searching for molecular cues that derive from the CNS and allow its direct communication with the immune system, we discovered a repertoire of CNS-derived endogenous regulatory (guardian) peptides presented on major histocompatibility complex (MHC) II molecules at the CNS borders. During homeostasis, a preponderance of these guardian peptides were found to be bound to MHC II molecules throughout the path of lymphatic drainage from the brain to its surrounding meninges and its draining cervical lymph nodes. With neuroinflammatory disease, however, the presentation of guardian peptides was diminished. By then boosting the presence of these guardian peptides, a population of suppressor CD4+ T cells could be expanded and CNS autoimmune disease significantly reduced. This unexpected discovery of CNS-derived autoimmune guardian peptides may be the molecular key adapting the CNS to receive information and to maintain continuous dialogue with the immune system while balancing overt autoreactivity. This sheds new light on how we conceptually think about and therapeutically target neuroinflammatory and neurodegenerative diseases.
B. Introduction:
[0202] For the longest time, the concept of immune privilege solidified a framework through which the interaction between the central nervous system (CNS) and the periphery was understood. Given the capacity of the CNS to tolerize the transplantation of autologous grafts or sarcomas1-3, it was widely presumed that the brain walls itself off from the peripheral immune system4. While the presence of physical barriers in the brain, together with its lack of lymphatic drainage, was thought to help the CNS sustain its immune-privileged nature, the rediscovery of a bona fide lymphatic network at the brain borders began breaking down this supposed disconnect from the periphery'50. In fact, juxtaposed with an immunologically frugal brain parenchyma, yvould be an immunologically rich niche of immune cells in the brain meninges, especially in the outermost membranous covering enveloping the CNS, the dura mater7-9.
[0203] Moreover, recent work has demonstrated the accessibility of CNS-derived antigens within these neuroimmune hubs, potentiating their presentation on major histocompatibility complex (MHC) II molecules to circulating self-reactive T cells7. Indeed, more than 30 years ago, the presence of circulating autoreactive T cells, even in healthy individuals, was reported10. Thus, while it is by now well accepted that autoreactive CD4+ T cells provoke autoimmune disease, their potentially beneficial roles across an array of diseases and in the normal functioning of the CNS have only more recently been appreciated11-13. This raises the enigma of how the CNS maintain immune tolerance while enhancing its visibility to the immune system. It is contemplated that the CNS seeks immunosurveillance yvhile actively providing immune cells with molecular cues to foster its tolerance to autoreactivity. [0204] To begin examining plausible molecular cues coming from the CNS to autoreactive CD4+ T cells, a thorough interrogation was performed of the CNS, its borders, and its draining lymph nodes for peptides bound to MHC II molecules. In establishing the MHC II peptidome of tire CNS, CNS-de rived endogenous, regulatory self-peptides, were identified, which were termed “guardian peptides". These self-antigens were found to be presented on MHC II molecules in homeostasis and evoked a population of suppressor CD4+ T cells that guarded the CNS from overt autoreactivity. Neuroinflammatory disease was marked by a paucity of guardian peptides and their restoration ameliorated the disease. Altogether, it is demonstrated here that the CNS deliberately presents itself through endogenous guardian peptides on MHC II molecules to communicate with and dampen autoreactive T-cell responses to secure CNS immunosurveillance.
C. Results: i) The MHC II peptidome uncovers the presentation of CNS-derived endogenous peptides
[0205] Driven by the hypothesis that the CNS may manipulate the presentation of antigens to communicate with the immune system during homeostasis, the study was begun by interrogating the repertoire of autoantigens presented on MHC II molecules. To address this question, mass spectrometry was performed to comprehensively evaluate the CNS and its associated tissues for peptides bound to MHC II molecules in C57BL/6J male mice (FIG. 13A). We found that CD1 lb+ CD11c- macrophages were the predominant MHC Il-expressing cells (antigen-presenting cells, APCs) in the brain, which also includes the leptomeninges, and the dura, whereas CD 19+ B cells were the main APCs in the deep cervical lymph nodes (dCLNs) and in tire superficial cervica lymph nodes (sCLNs) (FIGS. 16A-16E). Moreover, algorithmic predictions of MHC II- binding affinities utilizing three independent models14 16 identified a propensity for peptides with relatively weaker affinities to be bound on MHC II molecules in the brain. Whereas, further downstream along the path of lymphatic drainage their binding affinities increased, suggesting heterogeneity to the repertoire of peptides bound to MHC II molecules in different compartments (FIG. 16F).
[0206] We next took advantage of published transcriptomic data of mammalian brain17 to distinguish those peptides derived from proteins whose expression in the CNS was enriched. In doing so, we found that 18.0%, 12.7%, 2.5%, and 0.5% of total MHC Il-bound peptides in the brain, dura, dCLNs, and sCLNs, respectively, could be annotated as CNS-enriched (FIG. 13B). Analysis of the repertoire of CNS- enriched autoantigens across the different tissues disclosed an abundance of peptides derived from myelin basic protein (MBP) in the brain, dura, and dCLNs, and their apparent absence in the sCLNs (FIG. 13C). By comparing overlapping peptide sequences between tissues, we further validated the dCLNs as the major route for lymphatic drainage from the brain during homeostasis (FIG. 13D, FIG. 16G)18 19. Furthermore, alignment of tire amino acid sequences for the different MBP peptides identified across the brain, the dura and the dCLNs highlighted two distinct regions, namely MBP158-195 and MBP196-236 (FIG. 13E, FIG. 16H). The vast majority of MHC Il-bound MBP peptides were contained within the upstream MBP158-195 sequence, representing the only region draining to the dCLNs (FIG. 16H).
[0207] Experiments were repeated in C57BL/6J female and SJL/J male mice. As compared with C57BL/6J males, C57BL/6J females exhibited an abundance of MBP peptides bound to MHC II molecules in the brain as well as in the dura (FIG. 17A). Moreover, there was a similar overrepresentation of MHC II -bound peptides encompassing the MBPiss-iss region when compared to the MBP196.236 region in C57BL/6I females as well (FIG. 17B). Indeed, a considerable amount of identical CNS-enriched peptide sequences was found to be shared between C57BL/6I females and males, both in the brain and in the dural meninges (FIGS. 17C-17F). Upon evaluating the immunopeptidome of SJL/J male mice, we similarly observed MHC Il-bound peptides with relatively weaker binding affinities in the brain that increased towards the draining lymph nodes, further reiterating that a different character of endogenous antigens bound to MHC II molecules in the brain and dura when compared to the draining lymph nodes (FIG. 18A). Additionally, similar to C57BL/6J mice, we observed a heightened presentation of CNS-enriched self-antigens in the dural meninges (FIGS. 18B-18D). In contrast to C57BL/6J mice, however, the diversity of CNS-enriched proteins bound to MHC II molecules was increased in the SJL/J mice, which also displayed peptides derived from neurofilaments, microtubule- associated proteins and P-synuclein. among others (FIG. 18E). Despite this increased diversity, however, peptides comprising the same MBP158-195 and MBP196-236 regions as those found in C57BL/6J mice could still be identified in both the brain and the dural meninges of SJL/J mice (FIGS. 18E-18F). Collectively, these data demonstrated surprising conservation with respect to the presentation of MBP-derived peptides on MHC II molecules in the CNS (TABLE 11).
[0208] TABLE 11: Overlapping CNS-enriched peptide families identified in MHC II peptidomes ii) MBP peptides are non-encephalitogenic and suppress the immune response
[0209] The abundant presentation of MBP peptides captivated our interest in understanding their potential function in the CNS during homeostasis. Identified MBP peptides were screened by immunizing with them to assess their encephalitogenicity. Whereas immunization of rodents with myelin oligodendrocyte glycoprotein (MOG)s5 55 induced paralysis ty pical of experimental autoimmune encephalomyelitis (EAE)20 — a rodent model of multiple sclerosis (MS)21 22 — immunization with endogenous MBP peptides, such as MBP160-175, did not induce paralysis (FIG. 14A). As expected, these findings were in line with prior observations that the C57BL/6J strain is particularly resistant to MBP- induced EAE23. When we compared their draining lymph nodes following immunization, mice that had received MBPiso 175 peptides exhibited decreased cellularity relative to those immunized with MOG35 55 peptides (FIG. 14B). Moreover, MBP160-175, unlike MOG35-55, lacked an antigen-specific effector T cell response when assaying for IL-2 production by enzyme-linked immune absorbent sport (ELISpot) (FIG. 14C). To determine whether the low immunogenicity of MBP160-175 was specific to this particular peptide, we further tested different MBP peptides identified by our immunopeptidome. Immunization with MBP166-185, a peptide contained within the MBP158-195 region, also showed a lack of IL-2 production similar to that seen for MBPiso 175. In contrast, immunization with MBP192 -21s elicited an activated T cell response (FIGS. 19A-19B).
[0210] Based on the above findings, we wondered whether MBP peptides within the MBP158-195 region might serve to give rise to more suppressive T cells and thus suppress the immune response. To test that possibility, mice were co-immunized with encephalitogenic MOG35-55 peptide and our endogenous MBP peptides. Co-immunization of MOG35-55 peptide with either MBPiso-ns or MBPiss-iss, but not with MBP192.216, significantly attenuated the neuroinflammatory disease (FIG. 14D, FIG. 19C). To further ascertain that peptide specificity was entailed in the observed protection, the MBP160-175 sequence was directly modified by converting its arginine residues to citrulline. Citrullination has been implicated in a variety of autoimmune diseases, and studies have demonstrated that MBP itself becomes hypercitrullinated and that this correlates with increased disease severity in both MS and EAE24-25. We found that citrullinated MBP160-175, when co-immunized with MOG35-55, indeed no longer suppressed EAE, implying a specific immunomodulatory function for naked MBPiso-ns peptides in regulating neuroinflammation (FIG. 14D). Interestingly, the specific immunosuppression of neuroinflammatory disease with MBP160-175 peptides could be observed both in C57BL/6J females and in a completely different model of active EAE with SJL/J male mice (FIGS. 19D-19E).
[0211] Having observed a more pronounced protection with MBP160-175, studies then focused on this particular peptide to determine potential mechanisms for its immunosuppression. To begin investigating the T cell phenotype elicited in the presence of MBP160-175 in our co-immunization paradigm, T cells were individually sorted from draining lymph nodes of MOG35 55 only and of MOG35 55 + MBPiso 175 immunization groups, and then performed single-cell RNA sequencing (scRNA-seq) using the 10X Genomics Chromium -gene expression platform. Unsupervised clustering and dimensionality reduction was perfonned to project the cells into two dimensions using the uniform manifold approximation and projection (UMAP) algorithm. Differential gene expression resolved 15 distinct clusters by which CD4+ T cells and CD8+ T cells could be distinguished (FIG. 14E, FIG. 20A). By comparing the distribution of the identified clusters between the MOG35-55 and MOG35-55 + MBPieo-ns groups, we observed differences to regulatory Foxp3+ CD4+ T cells and an activated Cxcr3+ Ccl5+ CD8+ T cells, both of which have been described to have implications in autoimmune diseases, including MS26-28. Intriguingly, with MOG35-55 + MBPieu-ns co-immunization, the frequency of regulatory CD4+ T cells increased significantly, with an evident attenuation to the Cxcr3+ Ccl5+ CD8+ T cell cluster (FIG. 14F). These observations were in line with gene ontology analysis revealing upregulated pathways, such as negative regulation of T cell activation and proliferation as well as enhanced inhibitory cytokine (IL- 10 and TGF- b) production, in the MOG3555 + MBPiso 175 group (FIG. 14G). Altogether, scRNA-seq of both CD4+ and CD8+ T cells suggested that changes induced by MBP160-175 were already occurring in the draining lymph nodes prior to the onset of EAE, shaping the immune response towards that of suppression.
[0212] Having found MBP peptides on MHC II molecules, the study then focused on understanding its modulation of CD4+ T cell activity. Notably, concordant with the scRNA-seq data, the population of Foxp3+ CD4+ regulatory T cells remained heightened both in the draining lymph nodes and in the dCLNs at the onset of EAE with co-immunization of MBP160-175 relative to the MOG35-55 group (FIGS. 14H-14I. FIGS. 20B-20E). In the MBP160-175 co- immunized group, a significant increase in a CTLA-4+ Foxp3- CD4+ T cell population that expanded both in the dCLNs and in the spinal cord was observed (FIGS. 14H-14K). In fact, this same T cell population in the MBP160-175 co-immunized group also demonstrated increased CD39 expression in the spinal cord, suggesting its enhanced antiinflammatory activity through depletion of extracellular ATP (FIGS. 14J-14K)29. These cells, how ever, did not express PD-1 nor IL- 10 implying that they w ere distinct from previously reported Foxp3- non- conventional inhibitory T cells, such as T regulatory type 1 (Trl)30 and PD-lhi CD4+ T cells31 (FIG. 20B). Collectively, it is demonstrated that MBP guardian peptides reinforce both conventional regulatory and unconventional immunosuppressive CD4+ T cells to adequately guard against CNS autoimmune disease.
Hi) Therapeutic delivery of guardian MBP peptides regulates CNS autoimmunity
[0213] Tire above intriguing observations suggesting that endogenous guardian MBP peptides shape the immune response towards suppression led us to wonder whether their presentation w as altered in any way during neuroinflammation. To address this question the brain, dura, and spinal cord w as harvested from C57BL/6J male mice at the peak of EAE (day 16) to establish the MHC II peptidome during pathology (FIG. 15A). MHC II expression was found to be appreciably increased with neuroinflammation, thereby permitting adequate recovery of MHC Il-bound peptides using fewer mice (FIGS. 21A-21B). When compared the immunopeptidomes of EAE-induced and naive C57BL/6J mice, an obvious shift was observed in the repertoire of autoantigens presented on MHC II molecules (FIG. 15B): MHC Il-bound peptides derived from apolipoprotein E204 (Apoe) and prostaglandin D2 synthase (Ptgds), among others, were consistently found to be elevated in both the brain and the dura of EAE mice relative to naive controls. Not less intriguingly, with neuroinflammatory disease tire presentation of peptides within the MBP158 -195 region was found to be drastically diminished in both the brain and the dura, but this was not the case for the downstream MBP196-236 antigenic region (FIGS. 15B-15C). As a matter of fact, the only MBP-derived peptides that could be identified in either the brain or the spinal cord of EAE mice were those encompassing the MBP196236 region (FIG. 21C).
[0214] In a drive to understand potential causes of this surprising loss of MBP158-195 presentation on MHC II molecules, whether alterations to antigen processing could be responsible was investigated. By comparing the C-termini of peptides across EAE and naive mice, obvious changes to the cleavage profile for MHC Il-bound peptides were observed. In particular, EAE-induced mice had a significantly decreased predilection for basic residues at the C-terminus relative to naive mice (FIG. 2 ID). Knowing that myeloid cells in the CNS are major contributory APCs to both tire initiation and exacerbation of neuroinflammatory disease, a publicly available scRNA-seq dataset was utilized to understand alterations to their antigen-processing machinery in the context of EAE32. Where the vast majority of microglia lacked MHC II expression in homeostasis, neuroinflammation induced a disease-associated microglial phenotype with marked enhancement to MHC II expression and genes involved in antigen processing and presentation (FIGS. 2 IE and 21G)33. Moreover, we found that at the peak of EAE disease there was increased expression of peptidases, including cathepsins and caspases, among others (FIG. 2 IF). Such changes to the milieu of peptidases upon inflammation not only explicates shifts to the autoantigen repertoire presented on MHC 11 molecules but may also explain their diminishing capacity to present the MBPI SS-195 region.
[0215] Hie abundance of MBP158-195 presentation on MHC II molecules during homeostasis and together with its marked curtailment with acute neuroinflammation led us to wonder whether we could functionally employ these endogenous guardian MBP peptides to ameliorate CNS autoimmunity. By exploiting advancements in the therapeutic use of extracellular vesicles (EVs) and a recent demonstration of its ability to modulate EAE disease34,35, MBP160-175 peptides were encapsulated within EVs to test their efficacy as a potential antigen-specific immunotherapy (FIG. 22A). Surprinsingly, the introduction of EVs containing MBP160-175 into the CSF resulted in an evident expansion to the CTLA-4+ Foxp3- CD4+ suppressor T cells that we had observed earlier in the dura (FIGS. 15D-15E). Injection of empty EVs or of EVs with citrullinated MBP160-175 did not upregulate this CTLA-4+ Foxp3- CD4+ suppressor T cell population (FIG. 1 E), and indeed there were no differences in the frequencies of regulatory Foxp3+ CD4+ T cells across all tested conditions (FIG. 15E). Moreover, similar changes were consistently observed across multiple lymph nodes, including the dCLNs and sCLNs, but it did not affect the spleen (FIGS. 22B-22E). Therefore, intracistemal injection of EVs containing MBP160-175 affected both local and peripheral draining lymph nodes, reinforcing a specific population of suppressor CD4+ T cells. It was contemplated whether this would suffice to fend off CNS autoimmune disease. Congruously with our observations, in EAE-induced mice that had received EVs containing MBP160-175 peptides prior to the peak of EAE disease, CNS autoimmunity was significantly suppressed compared to controls receiving EVs with citrullinated MBPi60-i75 (FIG. 15F). Furthermore, extracellular vesicle-mediated delivery of MBP160-175 was necessary for its protection, as intracistemal injection of unencapsulated MBP160-175 peptides did not significantly alter disease progression (FIG. 22F).
[0216] In conclusion it was shown that by supplying the CSF with endogenous guardian MBP peptides the presence of suppressor CD4+ T cells can be boosted, which provides sufficient protection of the CNS against autoreactivity. These results lead us to further suggest that repleting a deficiency of guardian MBP peptides may broadly apply to the treatment of neuroinflammatory as well as of neurodegenerative disorders.
D. Discussion
[0217] In this study, the MHC II peptidome of the CNS during homeostasis is established, and endogenous guardian peptides are identified, which presented predominantly along the brain’s borders and within its draining lymph nodes. These guardian peptides guided the immune response towards suppression and specifically boosted a population of unconventional suppressor Foxp3- CD4+ T cells. Surprisingly, neuroinflammation resulted in a loss in the presentation of guardian peptides and supplementation of the CSF with these guardian peptides significantly ameliorated CNS autoimmune disease. This raises the possibility that the CNS seeks engagement of the adaptive immune system to acquire “immune privilege”.
[0218] Provided herein is the first account evidencing the direct binding of endogenous peptides sourcing from the CNS to MHC (e.g.. MHC II) molecules during homeostasis. We further spotlight an increased prevalence of MHC Il-bound MBP peptides pervading the meningeal lymphatic network, extending from the brain parenchyma to the draining cervical ly ph nodes. Studies in other systems have indeed demonstrated that tissue-specific antigens are presented on MHC II molecules during homeostasis in their respective draining lymph nodes for the skin and intestines36 as well as the pancreas37. Thus, we contemplate an intentional need for an organ system to provide an “image” of itself in order to calibrate the immune system towards self-tolerance38.
[0219] Potentially, these snapshots of self are relayed to autoreactive CD4+ T cells via their presentation on MHC II molecules. Strong support for this presumption comes from the present observation that endogenous guardian peptides identified herein can reinforce a specific population of unconventional suppressor CD4+ T cells in the CNS, thereby spurring on the immune response toward suppression. Therefore, tire CNS may have evolved a parallel mechanism that can manipulate the adaptive immune system to ensure CNS immune privilege, thereby adding an extra layer of protection to the physical barriers fortifying the CNS.
[0220] Such an exchange with the peripheral immune system comes with a risk. Indeed, in order to effectively mount an immune response against peripheral insults, the tissue shifts its focus to provide an “image” of the insult on MHC II molecules to appropriately tailor the adaptive immune response. Effectively, the immune response evolved to necessitate, on occasion, the displacement of self-antigens presented during homeostasis to resolve the more pressing issue, inflammation. This introduces vulnerabilities to the system - for while the immune response targets the insult, potential opportunities for pathologic self-reactivity through molecular mimicry arise39'41. Here, we demonstrate that the neuroinflammation induced by peripheral immunization alters antigen processing in the CNS. This drastically shifts the MHC II peptidome of the CNS and its borders, resulting in a loss in the presentation of these endogenous guardian peptides. These findings may shed new light onto the etiology of multiple sclerosis and of other autoimmune diseases, where peripheral infections alter the presentation of guardian peptides and thereby expose tire tissue to autoreactive attack.
[0221] Interestingly, in the context of neuroinflammation, reintroducing unmodified guardian peptides directly into the CSF attenuated CNS autoimmune disease. Thus, the findings of this study call attention to the feasibility of employing endogenous guardian peptides to rebalance and restore immune tolerance to the tissue. Furthermore, it emphasizes an attractive avenue along which these guardian peptides can be therapeutically administered as an antigen-specific immunotherapy for autoimmune disorders. In summary, we demonstrate that the CNS purposefully presents itself on MHC II molecules at the CNS borders (meninges), and that such presentation is critically important for its maintenance of immune tolerance.
E. Methods: i) Mice
[0222] C57BL/6J (WT: JAX000664) and SJL/J (WT; JAX000686) were purchased from the Jackson Laboratory; mice were maintained in standard housing conditions (12 hour light/dark cycle and provided with sterilized water and regular rodent chow ad libitum unless stated otherwise). Mice were allowed to acclimate for at least one week in the animal facility prior to the beginning of any experiment. Adult males and females between 8-12 weeks of age were primarily used for our studies unless stated otherwise. Sample sizes were determined on the basis of a power analysis in accordance with previously published experiments. Experimenters, where necessary, were blinded to experimental groups during both scoring and quantification. All experiments were approved by the Institutional Animal Care and Use Committee at the Washington University in St. Louis. ii) Single Cell Isolations
[0223] Brain (including leptomeninges), dural meninges, choroid plexus, spinal cord, lymph nodes (i.e. deep cervical lymph nodes, superficial cervical lymph nodes, and inguinal lymph nodes), and spleen isolations were performed following lethal i.p. injection of Euthasol. Subsequently, mice were perfused with transcardial perfusion with PBS containing heparin (0.025%); samples were collected into ice-cold RPMI and maintained on ice for the entirety of tissue collection. Spleens were removed from the surrounding tissues and placed on ice-cold RPMI (Gibco) until further use. Tire whole spleen was digested and mashed through a 70 pm cell strainer with a glass pestle and washed with 5 mL of RPMI. Cells were then centrifuged at 450g for 5 minutes. RBC lysis was performed with 1 mL of ACK lysis buffer (Quality’ Biological); cells were incubated for 2 minutes and then 2 mL of ice-cold PBS was supplemented to the sample. Samples were subsequently centrifuged at 450g for 5 minutes and lysed red blood cells were aspirated. The cell pellets were then resuspended in FACS buffer (2% BSA. 1 mM ethylenediamine acetic acid (EDTA), 20 mM HEPES) and kept on ice until further use. Lymph nodes were similarly digested and mashed through a 70 pm cell strainer, washed with 2 mL RPMI, centrifuged, resuspended in FACS buffer, and kept on ice until further use. Choroid plexi were removed from the brain; similarly, the dural meninges were peeled from the skull cap using Dumont #5 forceps (Fine Science Tools). Tire dural meninges were kept in ice-cold RPMI for the entirety of the tissue harvest. Dural meninges were then digested for 15 minutes at 37°C with constant agitation using 1 mL of prewarmed digestion buffer (RPMI-1640 medium with 2% FBS, 1 mg/mL Collagenase VIII, and 0.5 mg/mL DNase I). They were then filtered similarly through a 70 pm cell strainer and provided with 1 mL of complete medium (RPMI with 10% FBS) to neutralize the enzymes. Samples were then centrifuged at 450g for 5 minutes, resuspended in FACS buffer and kept on ice. Lastly, brains and spinal cords were harvested and placed in ice-cold RPMI for the entirety of the collection. Brains were mechanically dissociated using sterile surgical scalpels into ~1 mm3 cubes and digested with constant agitation in 2 mL of pre-warmed digestion buffer for 20 minutes at 37°C, triturated with a 10 mL serological pipette, digested for another 20 minutes, triturated with a 5 mL serological pipette, and digested for an additional 20 minutes. To remove myelin, a 1: 1 ratio of 22% bovine serum albumin (BSA) in PBS was added and centrifuged at 1000g for 10 minutes. Following centrifugation, the floating myelin layer was aspirated. Cell pellets were then neutralized with RPMI with 10% FBS to halt the enzymatic digestion. An additional 5 mL of RPMI with a glass pestle and washed with 5 mL of RPMI. Cells were then centrifuged at 450g for 5 minutes, resuspended in FACS buffer, and kept on ice until use.
Hi) Flow Cytometry
[0224] Single cell suspensions were acquired as described above in “single cell isolations,” and incubated initially with Zombie NIR (Biolegend) diluted 1:800 in ice-cold PBS for 15 minutes at 4°C for viability staining. Samples were then subsequently centrifuged, resuspended in FACS buffer containing anti-CD16/32 (Fc block; Biolegend) diluted 1: 100 in FACS buffer for 5 minutes. Cells were then stained for 30 minutes at 4°C for surface markers with antibodies diluted appropriately in FACS buffer. For surface staining only, samples were washed in FACS buffer and ran on the Aurora spectral flow cytometer (Cytek) then analyzed with Flow Jo software (Tree Star). Data processing and statistical analysis were performed using GraphPad Prism. For samples requiring intracellular staining, surface staining was performed first as described above. Then, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience): staining was performed for 30 minutes at 4°C with fluorescently conjugated antibodies against intracellular molecules. Similar to above, following staining, samples were washed with FACS buffer and ran on Aurora spectral flow cytometer. Data was analyzed with Flow Jo after which it was further processed and statistically analyzed by GraphPad Prism. For full list of antibodies used, see TABLE 12 below.
[0225] TABLE 12: Antibodies used in flow cytometry experiments iv) Isolation of the MHC-II peptidome
[0226] Tissues harvested from 110 8-10-week-old C57BL/6J male mice. 8-10-week-old C57BL/6J female mice, 8-10-week-old SJL/J male mice, as well as 20 8-10-week-old C57BL/6J male mice induced with EAE. Cells were isolated as described in '‘single cell isolations,” suspended in lysis buffer (40 mM MEGA 8, 40 mM MEGA 9, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM iodoacetamide, 20 pg ml— 1 leupeptin and Roche cOmplete Protease Inhibitor cocktail in phosphate-buffered saline), and rocked for 1 h at 4 °C. The cell lysate was spun in a centrifuge at 20,000g for 25 min at 4°C. To remove peptides that non-specifically bind to sepharose and/or immunoglobulin, the supernatant was first incubated with polyclonal mouse immunoglobulin G (Bio X Cell; 1.5 mg antibody per sample) bound to Sepharose 4B at 4 °C for 30 min. The flow through containing peptide-MHC II complexes was collected, added to a tube containing phosphate-buffered saline-washed sepharose conjugated to the anti-I-A antibody (Y-3P;
1.5 mg per sample), and incubated at 4°C overnight. The I-A- conjugated sepharose was applied to a column and washed four times as follows: 10 ml 150 mM NaCl and 20 mM Tris (pH 7.4); 10 ml 400 mM NaCl and 20 mM Tris (pH 7.4); 10 ml 150 mM NaCl and 20 mM Tris (pH 7.4); and 10 ml 20 mM Tris (pH 8.0). Peptides were then eluted with 10% acetic acid and dried by SpeedVac. Eluted peptides were passed over detergent removal spin columns (Pierce) to remove traces of remaining detergent and were further cleaned using C18 Spin Columns from Thermo Fisher Scientific (Pierce). v) Mass spectrometry
[0227] A Dionex UltiMate 1000 system (Thermo Fisher Scientific) was coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific) through an EASY-Spray ion source (Thermo Fisher Scientific). Prior to loading, peptide samples were reconstituted in 2% acetonitrile (ACN)/0.1% fonnic acid (17 pl). They were then subsequently loaded ( 15 pl, 15 pl min-1 ; 3 min) onto a trap column (100 pm x 2 cm; 5 pm Acclaim PepMap 100 C18; 50 °C), eluted (0.2 pl min-1 ) onto an EASY-Spray PepMap RSLC C18 column (2 pm; 50 cm x 75 pri ID; 50 °C; Thermo Fisher Scientific) and separated with the following gradient (all % buffer B (that is, 0.1% fomiic acid in acetonitrile)): 0-110 min: 2-22%; 110-120 min: 22- 35%; 120-130 min: 35-95%; 130-150 min: isocratic at 95%; 151-153 min: 95-2%; 153-171 min: isocratic at 2%. The spray voltage was 1,700 V, the ion transfer tube temperature was 275 °C and the RF lens was 30%. Mass spectrometry scans were acquired in profile mode and tandem mass spectrometry scans were acquired in centroid mode, for ions with charge states 2-7, with a cycle time of 1.5 s. Mass spcctromct ’ spectra were recorded from 375-1,500 Da at 120-K resolution (at m/z 200), and higher- energy collisional dissociation tandem mass spectrometry was triggered above a threshold of 2.0 x 104 . with quadrupole isolation (1.4 Da) at 15-K resolution and a collision energy of 30%. Dynamic exclusion was used (60 s), and monoisotopic precursor selection was on. vi) Mass spectrometry data analysis
[0228] Data files were uploaded to PEAKS Studio 10.6 (Bioinformatics Solutions) for processing, de novo sequencing and database searching. The sequences were searched against the UniProt Mouse database (downloaded 4 January 2022; 22,102 entries) with mass error tolerances of 10 ppm and 0.02 Da for parent and fragment, respectively, no enzyme specificity and oxidation (M), deamidation (NQ), and cysteine oxidation to cysteic acid as variable modifications. The Common Repository for Adventitious Proteins database (www.thegpm.org/crap/) was utilized to identify any contaminating proteins. FDR estimation was enabled. Peptides were filtered at a 5% FDR or a — lOlgP score of 15 (whichever was higher), and protein filtering was disabled by setting the protein -lOlgP score at 0 with one unique peptide and a requirement for significant peptides. For relative quantification of the different antigenic regions found in our peptidomes, we normalized peak areas to the total ion current (TIC), grouped overlapping peptide sequences into families, and summed their peak areas, where relevant. We then picked a common peptide family sequence for which similar peak areas could be identified to allow for comparisons betw een peptidomes of interest. “Relative abundances” were determined relative to the above-stated common peptide family sequence. vii) Experimental Autoimmune Encephalomyelitis (EAE)
[0229] EAE was induced in C57BL/6J mice by subcutaneous injection of MOG35-55 peptide (100 pg, CSBio) and/or different synthesized peptides including, MBP160-175, citrullinated MBPieo-ns. MBPiee-m. and MBPi 92-216 (GenScript) emulsified in Freund s adjuvant (Sigma .Aldnch) supplemented with 2 mg/mL of Mycobacterium tuberculosis (BD). Pertussis toxin (200 ng. List Biologicals) was injected i.p. on day 0 and day 2 following immunization with MOG and/or MBP. EAE was induced in SJL/J mice by subcutaneous injection of PLP139-151 (100 pg, Vivitide) and/or different synthesized peptides including, MBP160 175, neurofilament light polypeptide (NEFL)i6o-i73 (GenScript) emulsified in Freund's adjuvant (Sigma Aldrich) supplemented with 2 mg/mL of Mycobacterium tuberculosis (BD). Pertussis toxin (400 ng, List Biologicals) was injected i.p. on day 0 and day 2 following immunization with PLP and/or MBP and NEFM. For clinical evaluation, mice were scored daily: 0 = no clinical disease, 1 = limp tail, 2 = hindlimb weakness, 3 = hindlimb paralysis, 4 = partial front limb paralysis, 5 = moribund. For foil list of peptide sequences used, sec TABLE 13, below'.
[0230] TABLE 13: Peptide sequences used for immunizations viii) Ezyme-linked immune absorbent spot (ELI Spot)
[0231] Mice were subcutaneously immunized with different peptides, including MOG35 55, MBP160-175, MBP166-185, and MBP192-216, among others, emulsified in Freund’s adjuvant. 1 day prior to harvest, 96- well polyvinylidene fluoride membrane plates (Millipore) were coated with IL-2 capture antibodies (BD Biosciences). 7 days after immunization, cells were isolated from the draining lymph nodes and counted such that 5 x 105 cells would be loaded per well (each condition performed in triplicates). Cells were stimulated with their appropriate antigens and controls and incubated at 37°C in 5% CO2 for 24 hours. IL-2 secreting cells were identified with IL-2 detection antibodies (BD Biosciences) and plates were developed following manufacturer’s directions (BD Biosciences). Spots quantified with CTL ImmunoSpot S6 Universal machines and Professional 6.0.0 software. ix) Single-cell RNA Sequencing.
[0232] Inguinal lymph node T cells. C57BL/6J male mice were immunized either with MOG35 55 or MOG35.55 along with MBP160-175 (n = 3 per group) and 10 days later cells from the draining (inguinal) lymph nodes were harvested. Cells were isolated as described in “single cell isolations”, and sorted to capture those negative for DAPI (live cells), Ury 1.2 and TCRB to capture both CD4+ and CD8+ T cells for single-cell sequencing. Sample loading and library construction were performed using the 10X Genomics Chromium platform and Chromium Single Cell 3’ Library & Gel Bead Kit version 3 and libraries were sequenced on the Illumina NextSeq 500.
[0233] Reads were aligned to the mm 10 genome using the Cellranger software pipeline provided by lOx genomics. The resulting filtered gene by cell matrices of UMI counts were read into R using the read 1 OxCounts function from the Droplet Utils package. Cells were first filtered by manual removal of cells that had greater than 2.5 standard deviations from the mean of either total RNA count or unique features in both directions, or 3 standard deviations for percent of mitochondrial transcripts above the mean. Expression values for the final subset were then nonnalized using the scran and scater packages. The resulting log2 values were transformed to the natural log scale for compatibility with the Seurat (v3) pipeline42’44. The filtered and normalized matrix was used as input to the Seurat pipeline and cells were scaled across each gene, regressing out sequencing depth per cell, number of unique features, and percent mitochondrial reads, before the selection of the top 2,000 most highly variable genes using variance stabilizing transformation. Principal Components Analysis was conducted and an elbow plot was used to select components for UMAP analysis and clustering. Shared Nearest Neighbor (SNN) clustering optimized with the Louvain algorithm, as implemented by the Seurat FindClusters function was performed before manual annotation of clusters based on expression of canonical gene markers. Initial contamination in the form of clusters with markers of B cells or myeloid lineage as well as doublets were manually removed and the finalized subset of cells was scaled, underwent PCA, clustering, and annotation as described above. Statistical analysis of cluster proportions was done via a pennutation test with 1,000 instances followed by bootstrapping to produce a p-value and confidence interval.
[0234] For analysis of differentially expressed genes between conditions, CD4 and CD8 cells were subset based on sub-cluster identity and gene expression and each subset was filtered to include genes that had at least 4 transcripts in at least 4 cells. Then the top 2000 highly variable genes were determined and included for further analysis using the SingleCellExperiment modelGeneVar and getTopHVGs functions. After filtering, limma and edgeR were used to build a model and conduct differential expression testing with the ImFit, contrasts.fit, and eBayes functions. Results were then filtered using a Benjamini- Hochberg adjusted p-value threshold of less than 0.05 as statistically significant. Over representation enrichment analysis with Fisher’s Exact test was used to detennine significantly enriched Gene Ontology (GO) tenns (adj . p < 0.05) for the sets of significantly differentially expressed genes. For each gene set, genes were separated into up- and down regulated and separately45 the enrichGO function from the clusterProfiler package was used with a gene set size set between 10 and 500 genes and p-values adjusted using the Benjamini-Hochberg correction46. x) Jordao EAE microglia analysis
[0235] CSV files containing single-cell transcript counts were downloaded from the Gene Expression Omnibus accession GSE118948, read into R and converted to matrices. Blood and choroid plexus tissue samples were discarded and the remaining leptomeningeal and parenchymal perivascular space samples were manually filtered for quality control. Filtering was performed to remove cells with less than 200 unique genes, less than 1,000 or greater than 25,000 transcripts, and mitochondrial transcript percentage greater than 30 percent. Expression values were then normalized using the scran and scater packages. The resulting log2 values were transformed to the natural log scale for compatibility with the Seurat (v3) pipeline42-44. Initial contamination in the fonn of lowly sequenced clusters were manually removed and the finalized subset of cells was scaled, underwent PCA, clustering, and annotation as described above.
[0236] The filtered and normalized matrix was used as input to the Seurat pipeline and cells were scaled across each gene, regressing out sequencing depth per cell, number of unique features, and percent mitochondrial reads, before the selection of the top 2,000 most highly variable genes using variance stabilizing transformation. Principal Components Analysis was conducted and an elbow plot was used to
Ill select components for UMAP analysis and clustering. Shared Nearest Neighbor (SNN) clustering optimized with the Louvain algorithm, as implemented by the Seurat FindClusters function was performed before manual annotation of clusters based on expression of canonical gene markers. For differential gene expression analysis, samples were grouped into Naive, Pre-symptomatic (includes Pre- clinical 4d and Pre-clinical 8d from original data analysis) and EAE (includes onset and acutea from original data analysis). Microglia were subset and filtered to include genes that had at least 4 transcripts in at least 4 cells. Then the top 2000 highly variable genes were determined and included for further analysis using the SingleCellExperiment modelGeneVar and getTopHVGs functions. After filtering, limma and edgeR were used to build a model and conduct differential expression testing with the ImFit, contrasts. fit, and eBayes functions. Results were then filtered using a Benjamini -Hochberg adjusted p- value threshold of less than 0.05 as statistically significant. Over representation enrichment analysis with Fisher’s Exact test was used to determine significantly enriched Gene Ontology (GO) temis (adj . p < 0.05) for the sets of significantly differentially expressed genes. For each gene set, genes were separated into up- and down-regulated and separately45 the enrichGO function from the clusterProfiler package was used with a gene set size set between 10 and 500 genes and p-values adjusted using the Benjamini- Hochberg correction46. Up- or down-regulated peptidases were identified using the publicly available MEROPS database47. xi) Epitope prediction
[0237] To predict binding affinities of MHC Il-bound peptides for C57BL/6J mice (LAb haplotype) in the CNS and its associated tissues, we applied three independent models. These include: an algorithm developed by us, utilizing a hidden Markov model (HMM) and trained on the most recent immune epitope database (1EDB)14 as well as publicly available platfonns. such as nctMHCIIpan-4.015 and MHCnuggets16. Since it is not possible to directly combine scores from different models, we calculated the percentile rank of the result score for each peptide and model against a large decoy set of random natural peptides. The mean (or median) value of percentiles across all models was then used as the main source for the given antigen. We further represented these values as inverted percentile ranks such that larger values indicate peptides with increased likelihood to be a binder. The decoy set was generated from the mouse proteome and consists of about 1 x 106 peptide fragments. xii) Cell culture
[0238] The DC2.4 cell line was purchased from the American Type Culture Collection (ATCC). Cells were maintained by culturing in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin/streptomycin (Gibco) and 2 mM L-glutamine (Gibco). All cells were grown in an incubator set at 37°C in 5% CO2. xiii) Extracellular vesicles isolation and preparation
[0239] The DC2.4 cells were cultured as stated above in complete DMEM medium in T-75 flasks to 80- 90% confluency. Cells were thoroughly washed with dPBS prior to replacing the medium with serum depleted DMEM. They were further cultured overnight. Supernatants were collected next day and spun at varying speeds, including 1.000g for 10 mins to rid of any cell contaminants as well as 20,000g for 30 mins to rid of any smaller debris. The supernatant, at this point, was further filtered with a 0.22 pm filter and ultracentrifuged at 100,000g for 2 hours to pellet down extracellular vesicles (EVs). All spins were performed at 4°C. The pellet was resuspended in 500 pl PBS and visualized by either transmission electron microscope (JEOL JEM-1400, Phillips) or nanoparticle tracking analysis (ZetaView) to confirm their isolation, to measure their size, and to approximate the number of particles per milliliter
(mean ± SD). Isolated EVs were then sonicated with no antigen (empty EVs), MBP160-175, or citrullinated MBPieo 175 peptides at an amplitude equal to 20% for 6 cycles (30 s on, 2 mins off). Following this, they were allowed to recover in the incubator set at 37°C in 5% CO2 for 1 hour. To remove any unincorporated peptides and debris, we utilized the Exo-spinTM column and followed the manufacturer’s protocol (Cell Guidance Systems) to precipitate our EVs of interest. Similar as to above, EVs were assessed for successful purification by both TEM and nanoparticle tracking analysis. EVs were kept at - 80°C prior to intracistemal injection. xiv) Western blotting
[0240] Supernatant or pellet fraction after ultracentrifiigation of EVs resuspended in RIPA buffer.
Samples then appropriately diluted with 4x NuPAGETM LDS Sample Buffer (NP0007, Thermo Fisher Scientific) and boiled at 70°C for 10 mins. Samples subsequently loaded onto 15-well SDS-PAGE precasted gel (4-15% acrylamide gradient. #4561086, Bio-Rad) and electrophoresis performed in Bio-Rad cassette. Gel electrophoresis ran at 100 V for 1 h after which it was transferred to methanol activated PVDF membrane on ice for 2 h at 30 V. Membranes were then blocked with 5% (w/v) milk in trisbuffered saline, 0.1% Tween-20 (TBS-T) for 1 h at room temperature. Primary antibodies, including antimouse CD9 (1: 1000, 124802, BioLegend) and anti -mouse CD63 (1: 1000, 143901, BioLegend), were diluted in 5% milk in TBST after which it was used to stain the membrane overnight at 4°C. Membranes were then washed 3 times with TBS-T. Appropriate secondary antibodies diluted 1 : 10000 in 5% milk in TBS-T. This was used to stain the blot for 2 h at 4°C. Membranes washed again 3 times with TBS-T and developed thereafter with PierceTM ECL Western Blotting Substrate kit (32106, Thermo Fisher
Scientific). Hie blot was eventually imaged using the ChemiDocTM MP gel imaging system (Bio-Rad). xv) Intracisternal injection
[0241] Mice were anesthetized using ketamine/xylazine (100 mg/kg ketamine and 10 mg/kg xylazine). Their necks were shaved and cleaned appropriately with 70% iodine prior to placing their heads securely into the stereotactic frame. Ophthalmic solution was applied to prevent drying of their eyes prior to the procedure. Following this, a longitudinal incision of the skin was made at the back of their necks: the underlying muscles were retracted with hooks to expose the cistema magna. A 5pL Hamilton syringe with a 33-gauge needle was utilized to inject 5pL of PBS or extracellular vesicles (i.e. empty, MBP160- 175-containing, or citrullinated MBP160-175-containing) at an approximate rate of 2.5pL/minute, 105 vesicles/pL. To prevent backflow, the needle was not immediately retracted and kept within tire cistema magna for an additional minute following the injection. The skin was sutured, and the mice were maintained on a heating pad until fully awake. Following the surgery, the mice received a subcutaneous injection of ketoprofen (2.5 mg/kg). xvi) Statistical Methods
[0242] Sample sizes, where necessary, were selected based on a standard power calculation (a = 0.05, power of 0.8). Generally, statistical methods were not used to recalculate or to predetermine sample sizes. Variance was similar within comparable experimental groups. To ensure randomization, animals were selected from different cages but of the same experimental condition. Experimenters were kept blinded to the identity of groups prior to scoring up until data was collected and analyzed. Statistical tests utilized for each figure, where necessary; were deemed appropriate. Unpaired t-tests were performed when comparing between two independent groups. One-way ANOVA with appropriate multiple comparison test were performed when comparing between two independent groups. Two-way ANOVA with repeated measures and appropriate multiple comparisons test w ere performed when comparing betw een three or more independent groups requiring clinical symptom evaluation with consistent observations. Statistical analysis (data are always presented as mean ± s.e.m.) was performed using GraphPad Prism. xvii) Brain Data availability
[0243] Raw mass spectrometry data and peptide-spectrum matches were deposited in the MassIVE public proteomics repository (http://massive.ucsd.edu) with the identifier MSV000092643 and are available at ftp://massive.ucsd.edu/MSV000092643. Single-cell RNA sequencing is available at the Gene Expression Omnibus under accession GSE240691.
F. References for Example 4:
1. Shirai, Y. On the transplantation of the rat sarcoma in adult heterogenous animals. Jap Med World 1, 14-15 (1921).
2. Murphy, J. B. & Sturm, E. Conditions Determining the Transplantability of Tissues in the Brain. J Exp Med 38, 183-197 (1923).
3. Medawar, P. B. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 29, 58- 69 (1948).
4. Rustenhoven, J. & Kipnis, J. Bypassing the blood-brain barrier. Science 366, 1448-1449 (2019).
5. Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337-341 (2015).
6. Absinta, M. et al. Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. Elife 6 (2017).
7. Rustenhoven, J. ct al. Functional characterization of the dural sinuses as a ncuroimmunc interface. Cell 184, 1000-1016 el 027 (2021).
8. Rustenhoven, J. & Kipnis, J. Brain borders at the central stage of neuroimmunology. Nature 612, 417-429 (2022).
9. Castellani, G., Croese, T., Peralta Ramos, J. M. & Schwartz, M. Transforming the understanding of brain immunity. Science 380, eabo7649 (2023).
10. Ota, K. ct al. T-ccll recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346, 183-187 (1990).
11. Kipnis, J.. Gadani, S. & Derecki, N. C. Pro-cognitive properties of T cells. Nat Rev Immunol 12, 663-669 (2012).
12. Schwartz, M. & Raposo, C. Protective Autoimmunity: A Unifying Model for the Immune Network Involved in CNS Repair. Neuroscientist 20, 343-358 (2014).
13. Richards, D. M., Kyewski, B. & Feuerer, M. Re-examining the Nature and Function of SelfReactive T cells. Trends Immunol 37, 114-125 (2016).
14. Alspach, E. et al. MHC-II neoantigens shape tumour immunity and response to immunotherapy. Nature 574, 696-701 (2019). Reynisson, B. et al. Improved Prediction of MHC II Antigen Presentation through Integration and Motif Deconvolution of Mass Spectrometry MHC Eluted Ligand Data. J Proteome Res 19, 2304- 2315 (2020). Shao, X. M. et al. High-Throughput Prediction of MHC Class I and II Neoantigens with MHCnuggets. Cancer Immunol Res 8, 396-408 (2020). Sjostedt, E. et al. An atlas of the protein-coding genes in the human, pig, and mouse brain. Science 367 (2020). Cserr, H. F., Harling-Berg, C. J. & Knopf, P. M. Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance. Brain Pathol 2, 269-276 (1992). Louveau, A. et al. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci 21, 1380-1391 (2018). Mendel, I., Kerlero de Rosbo, N. & Ben-Nun, A. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells. Eur J Immunol 25, 1951-1959 (1995). Weiner, H. L. The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease? Ann Neurol 65, 239-248 (2009). Steinman, L. Immunology of relapse and remission in multiple sclerosis. Annu Rev Immunol 32, 257-281 (2014). Bernard, C. C. Experimental autoimmune encephalomyelitis in mice: genetic control of susceptibility. J Immunogenet 3, 263-274 (1976). Valesini, G. et al. Citrullination and autoimmunity. Autoimmun Rev 14, 490-497 (2015). Sospedra. M. & Martin. R. Immunology of multiple sclerosis. Annu Rev Immunol 23, 683-747 (2005). Balashov, K. E„ Rottman, J. B„ Weiner, H. L. & Hancock, W. W. CCR5(+) and CXCR3(+) T cells are increased in multiple sclerosis and their ligands MIP-lalpha and IP-10 are expressed in demyelinating brain lesions. Proc Natl Acad Sci U S A 96, 6873-6878 (1999). Mackay, C. R. CXCR3(+)CCR5(+) T cells and autoimmune diseases: guilty as charged? J Clin Invest 124, 3682-3684 (2014). Dominguez-Villar, M. & Hafler, D. A. Regulatory T cells in autoimmune disease. Nat Immunol 19, 665-673 (2018). Antonioli, L., Pacher, P., Vizi, E. S. & Hasko, G. CD39 and CD73 in immunity and inflammation. Trends Mol Med 19, 355-367 (2013). Roncarolo, M. G., Gregori, S., Bacchetta, R., Battaglia, M. & Gagliani, N. The Biology ofT Regulatory Type 1 Cells and Their Therapeutic Application in Immune -Mediated Diseases. Immunity 49, 1004-1019 (2018). Zappasodi, R. et al. Non-conventional Inhibitory CD4(+)Foxp3(-)PD-l(hi) T Cells as a Biomarker of Immune Checkpoint Blockade Activity. Cancer Cell 33, 1017-1032 el017 (2018). Jordao, M. J. C. et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363 (2019). Butovsky, O. & Weiner, H. L. Microglial signatures and their role in health and disease. Nat Rev Neurosci 19, 622-635 (2018). Wiklander, O. P. B., Brennan. M. A., Lotvall, J., Breakefield, X. O. & El Andaloussi, S. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med 11 (2019). Casella, G. et al. Oligodendrocyte -derived extracellular vesicles as antigen-specific therapy for autoimmune neuroinflammation in mice. Sci Transl Med 12 (2020). Fugmann, T., Sofron, A., Ritz, D., Bootz, F. & Neri, D. The MHC Class II Immunopeptidome of Lymph Nodes in Health and in Chemically Induced Colitis. J Immunol 198, 1357-1364 (2017). Wan, X. et al. Tire MHC-II peptidome of pancreatic islets identifies key features of autoimmune peptides. Nat Immunol 21, 455-463 (2020). Cohen, I. R. The cognitive paradigm and the immunological homunculus. Immunol Today 13, 490-494 (1992). Wucherpfennig, K. W. & Strominger, J. L. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80, 695-705 (1995). Lunemann, J. D. et al. EBNA1 -specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-gamma and IL-2. 1 Exp Med 205, 1763-1773 (2008). Lanz, T. V. et al. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603, 321-327 (2022). Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411-420 (2018). Lun, A. T., McCarthy, D. J. & Marioni, J. C. A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor. FlOOORes 5, 2122 (2016). 44. McCarthy, D. J., Campbell, K. R., Lun, A. T. & Wills, Q. F. Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33, 1179-1186 (2017).
45. Hong, G., Zhang, W., Li, H., Shen, X. & Guo, Z. Separate enrichment analysis of pathways for up- and downregulated genes. J R Soc Interface 11, 20130950 (2014).
46. Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284-287 (2012).
47. Rawlings, N. D. et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46, D624-D632 (2018).
5. Example 5: Assessment of Tubb 3, Map2, and Nefm as MHC guardian peptides in an EAE animal
[0244] Fragments of Map2, Neftn, and Tubb3 were evaluated as candidates for regulatory peptides (e.g., MHC guardian peptides) against CNS autoimmunity in an MOG33 55 induced EAE animal model, as described previously. Clinical EAE scores were tracked over 19 days post immunization with either MOG33-55 alone or MOG33-55 together with cither: one of four Tubb3 peptides; a Map2 peptide; or one of two Nefm peptides. Average clinical scores and statistics (two-way ANOVA) are shown in FIGS. 23A- 23G. Sequences of the tested peptides are shown below in TABLE 14.
[0245] TABLE 14: Tubb3, Map2, and Nefm peptides tested in a MOG33-55 induced EAE animal model

Claims

CLAIMS What is claimed is:
1. A pharmaceutical composition comprising one or more peptides and a pharmaceutically acceptable excipient, wherein each of the one or more peptides binds to a Major Histocompatibility Complex (MHC) molecule and is a fragment of a protein selected from TABLE Cl. or a modified fragment thereof.
2. The pharmaceutical composition of claim 1, wherein the protein is expressed at elevated levels in the central nervous system (CNS) relative to a control tissue.
3. The pharmaceutical composition of claim 1, wherein the protein is not expressed at elevated levels in the central nervous system (CNS), or is expressed at decreased levels in the CNS relative to a control tissue.
4. The pharmaceutical composition of claim 1. wherein each of the one or more peptides binds to an MHC class I molecule, to an MHC class II molecule, or to both.
5. The pharmaceutical composition of claim 1. wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or amodified sequence thereof.
6. Tire pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of
SEQ ID NOS:74, 75, 78, 91, 174, 176, 241, 242, 243. 244, 245. 246, 247. 248, 249. 250, 251. 252, 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof.
7. The pharmaceutical composition of claim 1. wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775, 3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774, 4775, 4776, 4777. 4778, 4779, 4780, 4781, 4782, 4783, 4784. 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519, 14520, 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, or a modified sequence thereof. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 8, or a modified sequence thereof. The pharmaceutical composition of claim 1, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS of TABLE 9, or a modified sequence thereof The pharmaceutical composition of claim 1. wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 10, or a modified sequence thereof. The pharmaceutical composition of claim 1. wherein each of the one or more peptides is a fragment of a myelin basic protein (MBP), tubulin beta 3 class 111 (TUBB3), or neurofilament medium polypeptide (NEFM). The pharmaceutical composition of claim 12, wherein each fragment of MBP independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278, 3784, 3785, 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796,
10752, 10753, 10754, 10755, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764,
10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777,
10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788. 10789, 10790,
10791, 10792, 10793, 10794, 10795, 10796, 10797. 10798, 10799, 10800, 10801. 10802, 10803,
10804, 10805, 10806, 10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816,
10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829,
10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545,
14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556, 14557, 14558,
14559, 14560, 14561, 14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582. 14583, 14584,
14585, 14586, 14587. 14588, 14589, 14590, 14591. 14592, 14593, 14594, 14595. 14596, 14610,
14611, 14612, and 14613. The pharmaceutical composition of claim 1, wherein at least one of the one or more peptides comprises or consists of the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO:5776). DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759). or a modified sequence thereof. The pharmaceutical composition of claim 1. further comprising one or more extracellular vesicles (EV) or liposomes enclosing the one or more peptides. Tire method of claim 15, wherein the pharmaceutical composition comprises one or more EV, and wherein the one or more EV is an exosome, a microvesicle, or an apoptotic body of a cell, optionally a human cell. The method of claim 15, wherein the pharmaceutical composition comprises one or more EV, and wherein the one or more EV is oligodendrocyte-derived, optionally from the cell line DC2.4. The method of claim 15, wherein the pharmaceutical composition comprises one or more EV. and wherein the one or more EV is synthetic. The pharmaceutical composition of any one of claims 1-18, for use in treating, preventing, or reversing a neuroinflammation-related disorder, an autoimmune neural disorder, or an acute CNS injury. Tire pharmaceutical composition of claim 19, wherein the neuroinflammation-related disorder or an autoimmune neural disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cold agglutinins, and disialosyl antibodies (CANOMAD), chronic meningitis, Behcet’s disease , Central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillar acidic protein (GFAP), Hashimoto's encephalitis, hypertrophic pachymeningitis, IgG4 associated neurological disease. Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurologic syndrome (PNS), Parkinson’s disease (PD), steroid responsive encephalopathy associated with autoimmune thyroiditis (SREAT). stiff person syndrome, Susac’s syndrome, and transverse myelitis. Tire pharmaceutical composition of claim 19, wherein the acute CNS injury is selected from the group consisting of spinal cory injury, traumatic brain injury, spinal cord injury, optic nerve injury, and stroke. A method of treating, preventing, or reversing a neuroinflammation -related disorder, autoimmune neural disorder, or an acute CNS injury in a subject in need thereof, comprising administering to the subject one or more peptides each independently comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof. Tire method of claim 22, wherein the neuroinflammation-related disorder or an autoimmune neural disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN), acute optic neuritis (AON), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cold agglutinins, and disialosyl antibodies (CANOMAD), chronic meningitis, Behcet’s disease , Central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillar acidic protein (GFAP), Hashimoto’s encephalitis, hypertrophic pachymeningitis, IgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetennined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurologic syndrome (PNS), Parkinson’s disease (PD), steroid responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff person syndrome, Susac’s syndrome, and transverse myelitis. The method of claim 22, wherein the acute CNS injury is selected from the group consisting of a spinal con injury, traumatic brain injury', spinal cord injury, optic nerve injury', and stroke. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:597, 770, 1231, 1232, 1709, 1748, 2297, 2324, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774, 3775,
3784, 3785, 3786, 3787, 3788, 4514, 4534, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551,
4552, 4553, 4554. 4555, 4556, 4557, 4558, 4642, 4643, 4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766. 4767. 4768, 4769, 4770, 4771, 4772, 4774, 4775. 4776, 4777, 4778, 4779, 4780,
4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794, 4795,
4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806, 4807, 4809, 4810, 4811,
4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828, 4829, 5067, 5439, 5574, 5575, 14519,
14520, 14521, 14522, 14523, 14524, 14525, and 14526, or a modified sequence thereof. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, or a modified sequence thereof. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 8, or a modified sequence thereof. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 9, or a modified sequence thereof. The method of claim 22, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 10, or a modified sequence thereof. The method of claim 22, wherein each of the one or more peptides is a fragment of a myelin basic protein (MBP), tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM). The method of claim 22, wherein each fragment of MBP independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 5776, 176. 278, 3784, 3785, 3786, 3787, 3788, 5438, 5439, 5773, 5774, 5775, 5777, 5778, 5779, 5780, 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754, 10755, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764, 10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777, 10778. 10779, 10780,
10781, 10782, 10783, 10784, 10785, 10786, 10787. 10788, 10789, 10790, 10791. 10792, 10793,
10794, 10795, 10796, 10797, 10798, 10799, 10800, 10801, 10802, 10803, 10804, 10805, 10806,
10807, 10808, 10809, 10810, 10811, 10812, 10813, 10814, 10815, 10816, 10817, 10818, 10819,
10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832,
10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842, 10843, 10844, 10845,
12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545, 14546. 14547, 14548,
14549, 14550, 14551, 14552, 14553, 14554, 14555. 14556, 14557, 14558, 14559. 14560, 14561,
14562, 14563, 14564, 14565, 14566, 14567, 14568, 14569, 14570, 14571, 14572, 14573, 14574,
14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587,
14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and
14613, or a modified sequence thereof The method of claim 22, wherein at least one of the one or more peptides comprises or consists of the amino acid sequence of, FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof. The method of claim 22, wherein the one or more peptides are enclosed in one or more extracellular vesicles (EV) or liposomes. The method of claim 34, wherein the pharmaceutical composition comprises one or more EV, and wherein the one or more EV is an exosome, a microvesicle, or an apoptotic body of a cell, optionally a human cell. The method of claim 34, wherein the pharmaceutical composition comprises one or more EV, and wherein the one or more EV is oligodendrocyte-derived. The method of claim 34, wherein the phannaceutical composition comprises one or more EV, and wherein the one or more EV is synthetic. Tire method of claim 21, wherein the one or more peptides arc administered to the subject by infusing or injecting into the cerebrospinal fluid (CSF) of the subject. One or more nucleic acids encoding one or more peptides independently comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-14619, or a modified sequence thereof. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of
SEQ ID NOS:74, 75, 78, 91, 174, 176, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252. 254, 278, 292, 294, 295, 296, 297, 298, 299, 300, 301, and 5438, or a modified sequence thereof. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:597, 770, 1231, 1232. 1709, 1748, 2297, 2324, 3765, 3766, 3767. 3768, 3769, 3770, 3771, 3772. 3773, 3774, 3775, 3784, 3785, 3786, 3787. 3788, 4514, 4534, 4544, 4545,
4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554, 4555, 4556, 4557, 4558, 4642, 4643,
4644, 4645, 4646, 4657, 4683, 4762, 4763, 4766, 4767, 4768, 4769, 4770, 4771, 4772, 4774,
4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782, 4783, 4784, 4785, 4786, 4787, 4788, 4789,
4790, 4791, 4792, 4793, 4794, 4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804,
4805, 4806, 4807, 4809, 4810, 4811, 4812, 4813, 4819, 4820, 4822, 4823, 4825, 4826, 4828,
4829, 5067, 5439. 5574, 5575, 14519. 14520, 14521, 14522, 14523, 14524. 14525, and 14526, or a modified sequence thereof. The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 7, or a modified sequence thereof, or a modified sequence thereof . The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 8, or a modified sequence thereof, or a modified sequence thereof . The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 9, or a modified sequence thereof, or a modified sequence thereof . The one or more nucleic acids of claim 39, wherein each of the one or more peptides independently comprises or consists of the amino acid sequence set forth in any one of the SEQ ID NOS of TABLE 10, or a modified sequence thereof, or a modified sequence thereof The one or more nucleic acids of claim 39, wherein each of the one or more peptides is a fragment of a myelin basic protein (MBP), tubulin beta 3 class III (TUBB3), or neurofilament medium polypeptide (NEFM). The one or more nucleic acids of claim 46, wherein each fragment of MBP independently comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS:5776, 176, 278. 3784, 3785, 3786, 3787, 3788. 5438. 5439. 5773, 5774, 5775, 5777, 5778. 5779. 5780. 5781, 5782, 5783, 5784, 5785, 5786, 5787, 5788, 5789, 5790, 5791, 5792, 5793, 5794, 5795, 5796, 10752, 10753, 10754, 10755, 10756, 10757, 10758, 10759, 10760, 10761, 10762, 10763, 10764,
10765, 10766, 10767, 10768, 10769, 10770, 10771, 10772, 10773, 10774, 10775, 10776, 10777,
10778, 10779, 10780, 10781, 10782, 10783, 10784, 10785, 10786, 10787, 10788. 10789, 10790,
10791, 10792, 10793, 10794, 10795, 10796, 10797. 10798, 10799, 10800, 10801. 10802, 10803,
10804, 10805. 10806. 10807, 10808, 10809, 10810. 10811, 10812, 10813, 10814. 10815. 10816,
10817, 10818, 10819, 10820, 10821, 10822, 10823, 10824, 10825, 10826, 10827, 10828, 10829, 10830, 10831, 10832, 10833, 10834, 10835, 10836, 10837, 10838, 10839, 10840, 10841, 10842,
10843, 10844, 10845, 12162, 14537, 14538, 14539, 14540, 14541, 14542, 14543, 14544, 14545,
14546, 14547, 14548, 14549, 14550, 14551, 14552, 14553, 14554, 14555, 14556. 14557, 14558,
14559, 14560, 14561, 14562, 14563, 14564, 14565. 14566, 14567, 14568, 14569. 14570, 14571,
14572, 14573, 14574, 14575, 14576, 14577, 14578, 14579, 14580, 14581, 14582, 14583, 14584, 14585, 14586, 14587, 14588, 14589, 14590, 14591, 14592, 14593, 14594, 14595, 14596, 14610, 14611, 14612, and 14613, or a modified sequence thereof. The one or more nucleic acids of claim 39, wherein at least one of the one or more peptides comprises or consists of the amino acid sequence of FLPRHRDTGILDSIGR (SEQ ID NO:5776), DTGILDSIGR (SEQ ID NO: 10755), or DTGILDSIGRFFSGDRGAPK (SEQ ID NO: 10759), or a modified sequence thereof. One or more vectors each comprising at least one of the one or more nucleic acids of any one of claims 39 to 48. The one or more vectors of claim 49, wherein the one or more vectors are viral vectors. The viral vectors of claim 50, which are derived from an adenovirus, adeno-associated virus, or retrovirus, optionally selected from a lentivirus. The one or more vectors of claim 49, wherein the vectors are non-viral vectors. Tire one or more nucleic acids of any one of claims 39-48, wherein the nucleic acids are mRNAs. The one or more nucleic acids of claim 53, wherein the mRNAs are non-replicating mRNAs, self- amplifying mRNAs (saRNAs), or circular RNAs (circRNA). The one or more nucleic acids of claim 54, wherein the mRNAs are non -replicating mRNAs, wherein each mRNA comprises a coding region encoding one of the one or more peptides, a first UTR, a second UTR, a 5’ cap, and a poly(A) tail, and wherein each coding region is flanked by the UTRs. The one or more nucleic acids of claim 55, wherein each 5" cap comprises a 7-methylguanosine (m7G) cap that is connected to a first nucleotide of each mRNA (N) by triphosphates (ppp) to form m7GpppNp. The one or more nucleic acids of claim 56, wherein each m7G cap comprises a methylated 2’-OH on the first nucleotide connecting the 5’ end of each mRNA to the cap (capl or m7GpppNimp). Tire one or more nucleic acids of claim 56, wherein each m7G cap comprises a methylated 2’-OH on the first nucleotide and a second nucleotide coimecting the 5’ end of each mRNA to the cap (cap2 or m7GpppNimpN2mp). The one or more nucleic acids of any one of claims 53-58, wherein each mRNA comprises one more modified nucleosides, optionally selected from the group consisting of pseudouridine (\|/), N1 -methylpseudouridine (m 1 \p). 5 -methoxyuridine (mo5U), 2-thiouridine (s2U), 5 -methylcytidine (m5C) and N6 -methyladenosine (m6A). The one or more nucleic acids of any one of claims 53-59, wherein the nucleic acids are encapsulated, optionally by lipid nanoparticles (LNPs), polyplexes, polymeric nanoparticles, lipopolyplexes (LPPs), or cationic polypeptides. The one or more nucleic acids of claim 60, encapsulated by LNPs. A pharmaceutical composition comprising the nucleic acids of any one of claims 39 to 48, the vectors of any one of claims 49 to 52, or the mRNAs of any one of claims 53-61. Tire pharmaceutical composition of claim 62, which is formulated for infusion or injection into cerebrospinal fluid (CSF). The pharmaceutical composition of any one of claims 62 to 63, for use in treating, preventing, or reversing a neuroinflammation-related disorder, autoimmune neural disorder, or an acute CNS injury in a subject in need thereof. The pharmaceutical composition for use according to claim 64, wherein the neuroinflammation- related disorder or an autoimmune neural disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute inflammatory demyelinating polyradiculoneuropathy (AIDP; Guillain-Barre syndrome (GBS)), acute motor axonal neuropathy (AMAN), acute motor and sensory axonal neuropathy (AMSAN). acute optic neuritis (AON), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS). autoimmune encephalitis (AIE), chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M paraprotein, cold agglutinins, and disialosyl antibodies (CANOMAD), chronic meningitis, Behcet's disease , Central nervous system (CNS) vasculitis, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS), glial fibrillar acidic protein (GFAP), Hashimoto’s encephalitis, hypertrophic pachymeningitis, IgG4 associated neurological disease, Lambert-Eaton myasthenic syndrome (LEMS), anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), Miller Fisher syndrome (MFS), monoclonal gammopathy of undetermined significance (MGUS), multifocal motor neuropathy (MMN), myasthenia gravis (MG), multiple sclerosis (MS), neuromyelitis optica (NMO), neurosarcoidosis, paraneoplastic neurologic syndrome (PNS), Parkinson’s disease (PD), steroid responsive encephalopathy associated with autoimmune thyroiditis (SREAT), stiff person syndrome, Susac’s syndrome, and transverse myelitis. The pharmaceutical composition for use according to claim 64, wherein the acute CNS injury is selected from a spinal cory injury, traumatic brain injury, spinal cord injury, optic nerve injury, or stroke.
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